Methods of treating cancer using anti human interleukin-1 receptor accessory protein (IL1 RAP) antibodies

ABSTRACT

The present invention provides an antibody or an antigen-binding fragment thereof with binding specificity for human interleukin-1 receptor accessory protein (IL1RAP) wherein the antibody or antigen-binding fragment is capable of inhibiting the binding of antibody ‘CAN04’ to human IL1RAP. The invention further provides the use of such antibodies or an antigen-binding fragments in the treatment and/or diagnosis of IL-1 associated diseases and conditions, including cancers such as acute myeloid leukemia and melanoma.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/255,585, filed Sep. 2, 2016, which is continuation application ofInternational Application No. PCT/GB2015/050647, which was filed on Mar.5, 2015, and claims priority to United Kingdom application no. GB1403875.6, which was filed on Mar. 5, 2014, all of which areincorporated by reference in their entirety.

FIELD OF INVENTION

The present invention relates to antibody-based agents for the treatmentand diagnosis of diseases and conditions associated with a IL-1biomarker (specifically, IL1RAP) and/or responsive to inhibition of IL-1signalling. In particular, there are provided antibody-based agents forthe treatment and diagnosis of cancers, including but not limited tochronic myeloid leukemia (CML), acute myeloid leukemia (AML) and cancersassociated with solid tumour formation (such as melanoma, lung cancer,and cancer of the breast).

BACKGROUND

Interleukin-1 Biology

Interleukin-1 (IL-1) is a potent pro-inflammatory cytokine that can beproduced by a variety of cell types, including mononuclear phagocytes,in response to infection and inflammation. The IL-1 family consists ofseven agonists, including IL-1α and IL-1β, and three naturally occurringreceptor antagonists, including the IL-1 receptor antagonist (IL-1Ra)(Dinarello, C A, Blood 1996, 87(6): 2095-147). Two IL-1 receptors, IL-1Rtype I and IL-1R type II, have been identified. Both receptors caninteract with all three forms of the IL-1 family molecules. IL-1RI isresponsible for mediating IL-1-induced cellular activation. However, theIL-1/IL-1RI complex cannot signal by itself, but is dependent onassociation with a second receptor chain, IL-1R Accessory Protein(IL1RAP) (Dinarello, C A, Blood 1996, 87(6): 2095-147). In contrast toIL-1RI, IL-1RII does not induce cellular activation upon binding to IL-1and thus IL-1RII functions as regulatory decoy receptor, leading to anet decrease in IL-1 available to bind to IL-1RI.

In addition to IL1-signaling, IL1RAP is critical for mediating theeffects of IL33, through the ST2/IL1RAP complex, and IL36, through theIL1Rrp2/IL1RAP complex (Garlanda et al, Immunity. 2013 Dec.12;39(6):1003-18)

IL-1 is a potent pro-inflammatory cytokine, which is induced at sites oflocal infection or inflammation and is involved in the regulation of avariety of physiological and cellular events (summarised in Dinarello CA, CHEST, 2000, 118: 503-508 and Dinarello, C A, Clin Exp Rheumatol,2002, 20(5 Suppl 27): S1-13). It is capable of activating several celltypes including leukocytes and endothelial cells. IL-1 induces andamplifies immunological responses by promoting the production andexpression of adhesion molecules, cytokines, chemokines and otherinflammatory mediators such as prostaglandin E₂ and nitric oxide (NO).As a consequence, local inflammation is amplified and sustained. Inaddition, the IL-1-induced production of inflammatory mediators resultsin fever, headache, hypotension and weight loss. Furthermore, IL-1 is ahematopoietic growth factor and has been shown to reduce the nadir ofleukocytes and platelets in patients during bone marrow transplantation.IL-1 has also been shown to promote angiogenesis by inducing theproduction of vascular endothelial growth factor, thereby promotingpannus formation and blood supply in rheumatic joints. Finally, IL-1 hasbeen shown to promote the bone and cartilage degradation in rheumaticdiseases.

The Role of IL-1 in Disease

IL-1 is implicated in a wide range of diseases and conditions rangingfrom gout to cancer (for reviews, see Dinarello et al., 2012, NatureReviews 11:633-652 and Dinarello, 2014, Mol. Med. 20(suppl. 1):S43-S58;the disclosures of which are incorporated herein by reference),including:

-   -   Joint, bone and muscle diseases, such as rheumatoid arthritis        and osteoarthritis;    -   Hereditary systemic autoinflammatory diseases, such as familial        Mediterranean fever;    -   Systemic autoinflammatory diseases, such as systemic juvenile        idiopathic arthritis and adult-onset Still's disease;    -   Common inflammatory diseases, such as gout and type 2 diabetes;    -   Acute-onset ischemic diseases, such as myocardial infarction;        and    -   Cancer.

A number of therapies for blocking IL-1 activity are approved and indevelopment. Targeting IL-1 began in 1993 with the introduction ofanakinra (Kineret; Amgen), a recombinant form of the naturally occurringIL-1 receptor antagonist (IL-1Ra), which blocks the activity of bothIL-1α and IL-1β; this therapeutic has since been used to demonstrate arole for IL-1 in numerous diseases (see above). Anakinra currentlydominates the field of IL-1 therapeutics owing to its good safetyrecord, short half-life and multiple routes of administration.Neutralising IL-1 with antibodies or soluble receptors has also provedto be effective, and the soluble decoy receptor rilonacept (Arcalyst;Regeneron) and the anti-IL-1β neutralizing monoclonal antibodycanakinumab (Ilaris; Novartis) have now been approved. Other therapeuticapproaches, including IL-1α neutralisation, a therapeutic vaccinetargeting IL-1β and a chimeric IL-1Ra, are in early clinical trials. Inaddition, orally active small-molecule inhibitors of IL-1 production,such as caspase 1 inhibitors, have been developed and are being tested

IL1RAP as a Biomarker for Neoplastic Disorders

Tumour biomarkers are endogenous proteins or metabolites whose amountsor modifications are indicative of tumour state, progressioncharacteristics, and response to therapies. They are present in tumourtissues or body fluids and encompass a wide variety of molecules,including transcription factors, cell surface receptors, and secretedproteins. Effective tumour markers are in great demand since they havethe potential to reduce cancer mortality rates by facilitating diagnosisof cancers at early stages and by helping to individualize treatments.During the last decade, improved understanding of carcinogenesis andtumour progression has revealed a large number of potential tumourmarkers. It is predicted that even more will be discovered in the nearfuture with the application of current technologies such as tissuemicroarrays, antibody arrays, and mass spectrometry.

Interleukin-1 receptor accessory protein (IL1RAP) has previously beenidentified as cell-surface biomarker associated with haematologicalneoplastic disorders such as chronic myeloid leukemia (CML), acutemyeloid leukemia (AML) and myelodysplastic syndromes (MDS) (for example,see WO 2011/021014 to Cantargia A B, Järås et al., 2010, Proc Natl AcadSci USA 107(37):16280-5, Askmyr et al., 2013, Blood. 121(18):3709-13 andBarreyro et al., 2012, Blood 120(6):1290-8, the disclosures of which areincorporated herein by reference). More recently, the usefulness ofIL1RAP as a diagnostic and therapeutic biomarker for solid tumours, suchas melanomas, has also been revealed (see WO 2012/098407 to CantargiaAB, the disclosures of which are incorporated herein by reference).

The present invention thus seeks to provide improved antibodies for usein the diagnosis and treatment of diseases and conditions associatedwith the IL1RAP biomarker and/or responsive to inhibition of IL-1 and/orIL-33 signalling

SUMMARY OF INVENTION

A first aspect of the invention provides an antibody or anantigen-binding fragment thereof (‘antibody polypeptides’) with bindingspecificity for interleukin-1 receptor accessory protein (‘IL1RAP’),wherein the antibody or antigen-binding fragment is capable ofinhibiting the binding of reference antibody ‘CAN04’ to human IL1RAP.

By “interleukin-1 receptor accessory protein”, “IL1RAP” and “IL1-RAP” wespecifically include the human IL1RAP protein, for example as describedin GenBank Accession No. AAB84059, NCBI Reference Sequence: NP_002173.1and UniProtKB/Swiss-Prot Accession No. Q9NPH3-1 (see also Huang et al.,1997, Proc. Natl. Acad. Sci. USA. 94 (24), 12829-12832, the disclosuresof which are incorporated herein by reference). IL1RAP is also known inthe scientific literature as IL1R3, C3orf13, FLJ37788, IL-1RAcP andEG3556.

Thus, the antibody polypeptides of the invention have specificity forIL1RAP. By “specificity” we mean that the antibody polypeptide iscapable of binding to IL1RAP in vivo, i.e. under the physiologicalconditions in which IL1RAP exists within the human body. Preferably, theantibody polypeptide does not bind to any other protein in vivo. Suchbinding specificity may be determined by methods well known in the art,such as ELISA, immunohistochemistry, immunoprecipitation, Western blotsand flow cytometry using transfected cells expressing IL1RAP.Advantageously, the antibody polypeptide is capable of bindingselectively to IL1RAP, i.e. it binds at least 10-fold more strongly toIL1RAP than to any other proteins.

By “reference antibody ‘CAN04’” we include an intact IgG antibodycomprising heavy and light chain variable regions having the amino acidsequences of SEQ ID NOS: 1 and 2, respectively. Unless otherwise stated,references herein to “CAN04” refer to an intact IgG antibody comprising(a) a heavy chain comprising a variable region as defined by SEQ ID NO:1and a constant region as defined by SEQ ID NO: 19, and (b) a light chaincomprising a variable region as defined by SEQ ID NO:2 and a constantregion as defined by SEQ ID NO: 18. Alternatively, a humanised versionof CAN04 (‘hCAN04’) may be used as the reference antibody. For example,the reference antibody may be an intact IgG antibody comprising (a) aheavy chain comprising a variable region as defined by any one of SEQ IDNOS:8 to 11 and a constant region as defined by SEQ ID NO: 19, and (b) alight chain comprising a variable region as defined by any one of SEQ IDNOS:15 to 17 and a constant region as defined by SEQ ID NO: 18.

As discussed below, the reference antibody ‘CAN04’ binds to domain 2 ofIL1RAP. Thus, it will be appreciated that the antibody or anantigen-binding fragment of the invention also binds to domain 2 ofIL1RAP

By “capable of inhibiting the binding of reference antibody ‘CAN04’ tohuman IL1RAP” we mean that the presence of the antibody polypeptides ofthe invention inhibits, in whole or in part, the binding of ‘CAN04’ tohuman IL1RAP. Such competitive binding inhibition can be determinedusing assays and methods well known in the art, for example usingBIAcore chips with immobilised IL1RAP and incubating with the referenceantibody ‘CAN04’ with and without an antibody polypeptide to be tested.Alternatively, a pair-wise mapping approach can be used, in which thereference antibody ‘CAN04’ is immobilised to the surface of the BIAcorechip, IL1RAP antigen is bound to the immobilised antibody, and then asecond antibody is tested for simultaneous IL1RAP-binding ability (see‘BIAcore Assay Handbook’, GE Healthcare Life Sciences, 29-0194-00 AA05/2012; the disclosures of which are incorporated herein by reference).

In a further alternative, competitive binding inhibition can bedetermined using flow cytometry. For example, to test whether a testantibody is able to inhibit the binding of the CAN04 reference antibodyto a cell surface antigen, cells expressing the antigen can bepre-incubated with the test antibody for 20 min before cells are washedand incubated with the reference CAN04 antibody conjugated to afluorophore, which can be detected by flow cytometry. If thepre-incubation with the test antibody reduces the detection of thereference CAN04 antibody in flow cytometry, the test antibody inhibitsthe binding of the reference antibody to the cell surface antigen. Ifthe antibody to be tested exhibits high affinity for IL1RAP, then areduced pre-incubation period may be used (or even no pre-incubation atall).

In a further alternative, competitive binding inhibition can bedetermined using an ELISA (e.g. as described in Example J).

By “an antibody or an antigen-binding fragment thereof” we includesubstantially intact antibody molecules, as well as chimaericantibodies, humanised antibodies, isolated human antibodies, singlechain antibodies, bispecific antibodies, antibody heavy chains, antibodylight chains, homodimers and heterodimers of antibody heavy and/or lightchains, and antigen-binding fragments and derivatives of the same.Suitable antigen-binding fragments and derivatives include, but are notnecessarily limited to, Fv fragments (e.g. single chain Fv anddisulphide-bonded Fv), Fab-like fragments (e.g. Fab fragments, Fab′fragments and F(ab)₂ fragments), single variable domains (e.g. V_(H) andV_(L) domains) and domain antibodies (dAbs, including single and dualformats [i.e. dAb-linker-dAb]). The potential advantages of usingantibody fragments, rather than whole antibodies, are several-fold. Thesmaller size of the fragments may lead to improved pharmacologicalproperties, such as better penetration of solid tissue. Moreover,antigen-binding fragments such as Fab, Fv, ScFv and dAb antibodyfragments can be expressed in and secreted from E. coli, thus allowingthe facile production of large amounts of the said fragments.

The phrase “an antibody or an antigen-binding fragment thereof” is alsointended to encompass antibody mimics (for example, non-antibodyscaffold structures that have a high degree of stability yet allowvariability to be introduced at certain positions). Those skilled in theart of biochemistry will be familiar with many such molecules, asdiscussed in Gebauer & Skerra, 2009, Curr Opin Chem Biol 13(3): 245-255(the disclosures of which are incorporated herein by reference).Exemplary antibody mimics include: affibodies (also called Trinectins;Nygren, 2008, FEBS J, 275, 2668-2676); CTLDs (also called Tetranectins;Innovations Pharmac. Technol. (2006), 27-30); adnectins (also calledmonobodies; Meth. Mol. Biol., 352 (2007), 95-109); anticalins (DrugDiscovery Today (2005), 10, 23-33); DARPins (ankyrins; Nat. Biotechnol.(2004), 22, 575-582); avimers (Nat. Biotechnol. (2005), 23, 1556-1561);microbodies (FEBS J, (2007), 274, 86-95); peptide aptamers (Expert.Opin. Biol. Ther. (2005), 5, 783-797); Kunitz domains (J. Pharmacol.Exp. Ther. (2006) 318, 803-809); affilins (Trends. Biotechnol. (2005),23, 514-522); affimers (Avacta Life Sciences, Wetherby, UK).

Also included within the scope of the invention are chimeric T-cellreceptors (also known as chimeric T cell receptors, chimericimmunoreceptors, and chimeric antigen receptors or CARs) (see Pule etal., 2003, Cytotherapy 5(3):211-26, the disclosures of which areincorporated herein by reference). These are engineered receptors, whichgraft an arbitrary specificity onto an immune effector cell. Typically,CARs are used to graft the specificity of a monoclonal antibody onto a Tcell; with transfer of their coding sequence facilitated by retroviralvectors. The most common form of such molecules is fusions comprising asingle-chain variable fragment (scFv) derived from a monoclonal antibodyfused to CD3-zeta transmembrane and endodomain. When T cells expressthis fusion molecule, they recognize and kill target cells that expressthe transferred monoclonal antibody specificity.

Persons skilled in the art will further appreciate that the inventionalso encompasses modified versions of antibodies and antigen-bindingfragments thereof, whether existing now or in the future, e.g. modifiedby the covalent attachment of polyethylene glycol or another suitablepolymer (see below).

Methods of generating antibodies and antibody fragments are well knownin the art. For example, antibodies may be generated via any one ofseveral methods which employ induction of in vivo production of antibodymolecules, screening of immunoglobulin libraries (Orlandi. et al, 1989.Proc. Natl. Acad. Sci. U.S.A. 86:3833-3837; Winter et al., 1991, Nature349:293-299, the disclosures of which are incorporated herein byreference) or generation of monoclonal antibody molecules by cell linesin culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the Epstein-Barrvirus (EBV)-hybridoma technique (Kohler et al., 1975. Nature256:4950497; Kozbor et al., 1985. J. Immunol. Methods 81:31-42; Cote etal., 1983. Proc. Natl. Acad. Sci. USA 80:2026-2030; Cole et al., 1984.Mol. Cell. Biol. 62:109-120, the disclosures of which are incorporatedherein by reference).

Suitable methods for the production of monoclonal antibodies are alsodisclosed in “Monoclonal Antibodies: A manual of techniques”, H Zola(CRC Press, 1988, the disclosures of which are incorporated herein byreference) and in “Monoclonal Hybridoma Antibodies: Techniques andApplications”, J G R Hurrell (CRC Press, 1982, the disclosures of whichare incorporated herein by reference).

Likewise, antibody fragments can be obtained using methods well known inthe art (see, for example, Harlow & Lane, 1988, “Antibodies: ALaboratory Manual”, Cold Spring Harbor Laboratory, New York, thedisclosures of which are incorporated herein by reference). For example,antibody fragments according to the present invention can be prepared byproteolytic hydrolysis of the antibody or by expression in E. coli ormammalian cells (e.g. Chinese hamster ovary cell culture or otherprotein expression systems) of DNA encoding the fragment. Alternatively,antibody fragments can be obtained by pepsin or papain digestion ofwhole antibodies by conventional methods.

The antibodies of the invention are defined by reference to the variableregions of a murine-derived antibody, designated ‘CAN04’, whichcomprises:

(a) a heavy chain variable region having theamino acid sequence of SEQ ID NO: 1: [SEQ ID NO: 1]Q V Q L Q Q S G P E L L K P G A S V K I S C KA S G Y A F S S S W M N W V K Q R P G K G L EW I G R I Y P G D G N T H Y S G K F K G K A TL T A D K S S S I A Y M Q L S S L T S E D S AV Y F C G E G Y L D P M D Y W G Q G T S V T V S S and(b) a light chain variable region having theamino acid sequence of SEQ ID NO: 2: [SEQ ID NO: 2]D I Q M T Q T T S S L S A S L G D R V T I S CS A S Q G I N N Y L N W Y Q Q K P D G T V K LL I H Y T S G L H A G V P S R F S G S G S G TD Y S L T I S N L E P E D V A T Y Y C Q Q Y SI L P W T F G G G T K L E I K R

The term “amino acid” as used herein includes the standard twentygenetically-encoded amino acids and their corresponding stereoisomers inthe ‘D’ form (as compared to the natural ‘L’ form), omega-amino acidsand other naturally-occurring amino acids, unconventional amino acids(e.g. α,α-disubstituted amino acids, N-alkyl amino acids, etc.) andchemically derivatised amino acids (see below).

When an amino acid is being specifically enumerated, such as “alanine”or “Ala” or “A”, the term refers to both L-alanine and D-alanine unlessexplicitly stated otherwise. Other unconventional amino acids may alsobe suitable components for polypeptides of the present invention, aslong as the desired functional property is retained by the polypeptide.For the peptides shown, each encoded amino acid residue, whereappropriate, is represented by a single letter designation,corresponding to the trivial name of the conventional amino acid.

In one embodiment, the antibody polypeptides as defined herein compriseor consist of L-amino acids.

It will be appreciated by persons skilled in the art that any intact IgGantibody comprising the above variable regions may be used as thereference antibody to identify antibody polypeptides of the inventionthat competitively inhibit CAN04 binding to IL1RAP.

Thus, in one embodiment, the CAN04 antibody used as a reference todetermined competitive binder is an intact IgG antibody comprising:

-   -   (a) a heavy chain comprising a variable domain of SEQ ID NO:1        grafted on to a murine IgG1 or IgG2a constant region    -   (b) a light chain comprising a variable domain of SEQ ID NO:2        grafted on to a murine kappa constant region.

Alternatively, the reference antibody may be a chimeric, intact IgGantibody comprising:

-   -   (a) a heavy chain comprising a variable domain of SEQ ID NO:1        grafted on to an human IgG1 constant region (for example, as        encoded by the pFUSEss-CHIg-hG1 vector InvivoGen, San Diego,        USA)    -   (b) a light chain comprising a variable domain of SEQ ID NO:2        grafted on to a human kappa constant region (for example, as        encoded by the pFUSE2ss-CLIg-hk vector InvivoGen, San Diego,        USA).

Competitive binding typically arises because the test antibody binds at,or at least very close to, the epitope on the antigen to which binds thereference antibody (in this case, CAN04). However, it will beappreciated by persons skilled in the art that competitive binding mayalso arise by virtue of steric interference; thus, the test antibody maybind at an epitope different from that to which the reference antibodybinds but may still be of sufficient size or configuration to hinder thebinding of the reference antibody to the antigen.

The antibodies and antigen-binding fragments of the present inventionwere identified after extensive screening of a large number ofanti-IL1RAP antibodies, on the basis of exhibiting properties that makethem particularly suitable as diagnostic and therapeutic agents forcancer.

Thus, in one embodiment, the antibody or antigen-binding fragmentexhibits one or more of the following properties:

-   -   (a) a binding affinity (K_(D)) for human IL1RAP of 200 pM or        greater, i.e. the K_(D)≤200 pM (for example, as determined in        Example A);    -   (b) cross-reactivity with IL1RAP from Macaca fascicularis (for        example, as determined in Example D);    -   (c) an inhibitory action on IL1 signalling (IL-1α and/or IL-1β;        for example, as determined in Example E);    -   (d) capability of inducing antibody-dependent cell-mediated        cytotoxicity (ADCC) in one or more cancer cell lines (such as a        CML, ALL and/or melanoma cell lines) (for example, as determined        in Example F); and/or    -   (e) capability of internalisation upon binding to one or more        cancer cell lines (such as a CML, AML and/or melanoma cell line)        (for example, as determined in Example G).

Advantageously, the antibody or antigen-binding fragment exhibits all ofthe above properties.

In an alternative embodiment, the antibody or antigen-binding fragmentexhibits one or more of properties (a), (b), (c) and (e) above, but isnot capable of inducing ADCC.

In one embodiment, the antibody or antigen-binding fragment is capableof binding to an epitope on the extracellular domain of IL1RAP whichoverlaps, at least in part, with the epitope on IL1RAP to whichreference antibody CAN04 is capable of binding. Thus, the antibody orantigen-binding fragment may be capable of binding to an epitope locatedat/within domain 2 of IL1RAP (see Wang et al., 2010, Nature Immunology,11:905-912, the disclosures of which are incorporated herein byreference), i.e. within amino acids 135 to 234 of IL1RAP (see AccessionNo. Q9NPH3 within UniProtKB/Swiss-Prot). For example, the epitope towhich the antibody or antigen-binding fragment may be located withinamino acids 135 to 154, 155 to 174, 175 to 194, 195 to 214 or betweenamino acids 215 to 234 of IL1RAP. However, it will be appreciated thatthe epitope may be non-linear.

In one embodiment, the antibody polypeptide of the invention comprisesor consists of an intact antibody (such as an IgG1 antibody).

In an alternative embodiment, the antibody polypeptide of the inventioncomprises or consists of an antigen-binding fragment selected from thegroup consisting of Fv fragments (e.g. single chain Fv anddisulphide-bonded Fv), Fab-like fragments (e.g. Fab fragments, Fab′fragments and F(ab)₂ fragments) and domain antibodies (e.g. single V_(H)variable domains or V_(L) variable domains).

In a further embodiment, as discussed above, the polypeptide of theinvention comprises or consists of an antibody mimic selected from thegroup comprising or consisting of affibodies, tetranectins (CTLDs),adnectins (monobodies), anticalins, DARPins (ankyrins), avimers, iMabs,microbodies, peptide aptamers, Kunitz domains and affilins.

In a preferred embodiment, the antibody or antigen-binding fragmentthereof according to the first aspect of the invention comprises a heavychain variable region comprising the following CDRs:

-   -   a) GYAFSSS [SEQ ID NO: 3] or an amino acid sequence having at        least 60% sequence identity therewith, for example at least 70%,        80%, or 90% sequence identity;    -   b) YPGDGN [SEQ ID NO: 4] or an amino acid sequence having at        least 60% sequence identity therewith, for example at least 70%,        80%, or 90% sequence identity; and/or    -   c) GYLDPMDY [SEQ ID NO: 5] or an amino acid sequence having at        least 60% sequence identity therewith, for example at least 70%,        80%, or 90% sequence identity.

Thus, the antibody or antigen-binding fragment thereof may comprise aheavy chain variable region comprising the CDRs of SEQ ID NOs 3, 4 and5.

For example, the antibody or antigen-binding fragment thereof maycomprise a heavy chain variable region having the amino acid sequence ofthe corresponding region of the CAN04 reference antibody, i.e. SEQ IDNO:1.

However, it will be appreciated that a low level of mutation (typically,just one or two amino acids) within a CDR sequence may be toleratedwithout loss of the specificity of the antibody or antigen-bindingfragment for IL1RAP.

Percent identity can be determined by, for example, the LALIGN program(Huang and Miller, Adv. Appl. Math. (1991) 12:337-357, the disclosuresof which are incorporated herein by reference) at the Expasy facilitysite (http://www.ch.embnet.org/software/LALIGN_form.html) using asparameters the global alignment option, scoring matrix BLOSUM62, openinggap penalty −14, extending gap penalty −4. Alternatively, the percentsequence identity between two polypeptides may be determined usingsuitable computer programs, for example the GAP program of theUniversity of Wisconsin Genetic Computing Group and it will beappreciated that percent identity is calculated in relation topolypeptides whose sequence has been aligned optimally.

The alignment may alternatively be carried out using the Clustal Wprogram (as described in Thompson et al., 1994, Nucl. Acid Res.22:4673-4680, which is incorporated herein by reference). The parametersused may be as follows:

-   -   Fast pair-wise alignment parameters: K-tuple (word) size; 1,        window size; 5, gap penalty; 3, number of top diagonals; 5.        Scoring method: x percent.    -   Multiple alignment parameters: gap open penalty; 10, gap        extension penalty; 0.05.    -   Scoring matrix: BLOSUM.

Alternatively, the BESTFIT program may be used to determine localsequence alignments.

In a further preferred embodiment, the antibody or antigen-bindingfragment thereof according to the first aspect of the inventioncomprises a heavy chain variable region comprising the following CDRs:

-   -   a) GYAFSSSWMN [SEQ ID NO: 6] or an amino acid sequence having at        least 60% sequence identity therewith, for example at least 70%,        80%, or 90% sequence identity;    -   b) RIYPGDGNTHYSGKFKG [SEQ ID NO: 7] or an amino acid sequence        having at least 60% sequence identity therewith, for example at        least 70%, 80%, or 90% sequence identity; and    -   c) GYLDPMDY [SEQ ID NO: 5] or an amino acid sequence having at        least 60% sequence identity therewith, for example at least 70%,        80%, or 90% sequence identity.

For example, the antibody polypeptide may comprise a heavy chainvariable region comprising the CDRs of SEQ ID NOs 6, 7 and 5.

As indicated above, the CAN04 reference antibody is a murine antibody.However, the component heavy and light chains of this antibody may behumanised in order to produce antibody polypeptides more suitable foruse in humans, e.g. due to their reduced immunogenicity. For example,the CDRs of SEQ ID NOs 3, 4 and 5 (or the CDRs of SEQ ID NOs 6, 7 and 5)may be engrafted into a human variable region framework.

It will be appreciated by persons skilled in the art that for humantherapy, human or humanised antibodies are preferably used. Humanisedforms of non-human (e.g. murine) antibodies are genetically engineeredchimaeric antibodies or antibody fragments having preferablyminimal-portions derived from non-human antibodies. Humanised antibodiesinclude antibodies in which complementary determining regions of a humanantibody (recipient antibody) are replaced by residues from acomplementary determining region of a non-human species (donor antibody)such as mouse, rat of rabbit having the desired functionality. In someinstances, Fv framework residues of the human antibody are replaced bycorresponding non-human residues. Humanised antibodies may also compriseresidues which are found neither in the recipient antibody nor in theimported complementarity determining region or framework sequences. Ingeneral, the humanised antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the complementarity determining regions correspondto those of a non-human antibody and all, or substantially all, of theframework regions correspond to those of a relevant human consensussequence. Humanised antibodies optimally also include at least a portionof an antibody constant region, such as an Fc region, typically derivedfrom a human antibody (see, for example, Jones et al., 1986. Nature321:522-525; Riechmann et al., 1988, Nature 332:323-329; Presta, 1992,Curr. Op. Struct. Biol. 2:593-596, the disclosures of which areincorporated herein by reference).

Methods for humanising non-human antibodies are well known in the art.Generally, the humanised antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues, often referred to as imported residues, aretypically taken from an imported variable domain. Humanisation can beessentially performed as described (see, for example, Jones et al.,1986, Nature 321:522-525; Reichmann et al., 1988. Nature 332:323-327;Verhoeyen et al., 1988, Science 239:1534-15361; U.S. Pat. No. 4,816,567,the disclosures of which are incorporated herein by reference) bysubstituting human complementarity determining regions withcorresponding rodent complementarity determining regions. Accordingly,such humanised antibodies are chimaeric antibodies, whereinsubstantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanised antibodies may be typically human antibodies inwhich some complementarity determining region residues and possibly someframework residues are substituted by residues from analogous sites inrodent antibodies.

Human antibodies can also be identified using various techniques knownin the art, including phage display libraries (see, for example,Hoogenboom & Winter, 1991, J. Mol. Biol. 227:381; Marks et al., 1991, J.Mol. Biol. 222:581; Cole et al., 1985, In: Monoclonal antibodies andCancer Therapy, Alan R. Liss, pp. 77; Boerner et al., 1991. J. Immunol.147:86-95, the disclosures of which are incorporated herein byreference).

Thus, the antibody or antigen-binding fragment thereof of the inventionmay be humanised, for example it may comprise a heavy chain variableregion having one of the following amino acid sequence of any one of SEQID NOs: 8 to 11 or an amino acid sequence having at least 90% sequenceidentity therewith:

a) [SEQ ID NO: 8] Q V Q L V Q S G A E V K K P G S S V K V S C KA S G Y A F S S S W M N W V R Q A P G Q G L EW M G R I Y P G D G N T H Y A Q K F Q G R V TL T A D K S T S T A Y M E L S S L R S E D T AV Y Y C G E G Y L D P M D Y W G Q G T L V T V S S; b) [SEQ ID NO: 9]Q V Q L V Q S G A E V K K P G S S V K V S C KA S G Y A F T S S W M N W V R Q A P G Q G L EW M G R I Y P G D G N T H Y A Q K F Q G R V TL T A D K S T S T A Y M E L S S L R S E D T AV Y Y C G E G Y L D P M D Y W G Q G T L V T V S S; c) [SEQ ID NO: 10]Q V Q L V Q S G A E V K K P G S S V K V S C KA S G Y T F T S S W M N W V R Q A P G K G L EW M G R I Y P G D G Q T H Y A Q K F Q G R V TL T A D K S T S T A Y M E L S S L R S E D T AV Y Y C G E G Y L D P M D Y W G Q G T L V T V S S; or d) [SEQ ID NO: 11]Q V Q L V Q S G A E V K K P G S S V K V S C KA S G Y T F T S S W M N W V R Q A P G K G L EW M G R I Y P G D G Q T H Y A Q K F Q G R V TI T A D K S T S T A Y M E L S S L R S E D T AV Y Y C G E G Y L D P M D Y W G Q G T L V T V S S.

For example, the antibody or antigen-binding fragment thereof maycomprise a heavy chain variable region having an amino acid sequence ofany one of SEQ ID NOs: 8 to 11.

In a related preferred embodiment, the antibody or antigen-bindingfragment thereof comprises a light chain variable region comprising thefollowing CDRs:

-   -   a) SASQGINNYLN [SEQ ID NO: 12] or an amino acid sequence having        at least 60% sequence identity therewith, for example at least        70%, 80%, or 90% sequence identity;    -   b) YTSGLHA [SEQ ID NO: 13] or an amino acid sequence having at        least 60% sequence identity therewith, for example at least 70%,        80%, or 90% sequence identity; and/or    -   c) QQYSILPWT [SEQ ID NO: 14] or an amino acid sequence having at        least 60% sequence identity therewith, for example at least 70%,        80%, or 90% sequence identity.

Thus, the antibody or antigen-binding fragment thereof may comprise alight chain variable region comprising the CDRs of SEQ ID NOs 12, 13 and14.

For example, the antibody or antigen-binding fragment thereof maycomprise a light chain variable region having the amino acid sequence ofthe corresponding region of the murine CAN04 reference antibody, i.e.SEQ ID NO:2.

As in the case of the heavy chain variable region detailed above, itwill be appreciated that the light chain variable region of the antibodypolypeptide of the invention may be humanised in order to produce agentsmore suitable for use in humans. For example, the CDRs of SEQ ID NOs 12,13 and 14 may be engrafted into a human variable region framework.

Thus, the antibody or antigen-binding fragment thereof may comprise alight chain variable region which comprises or consists of the aminoacid sequence of any one of SEQ ID NOs: 15 to 17 or an amino acidsequence having at least 90% sequence identity therewith:

a) [SEQ ID NO: 15] D I Q M T Q S P S S L S A S V G D R V T I T CS A S Q G I N N Y L N W Y Q Q K P G K A P K LL I H Y T S G L H A G V P S R F S G S G S G TD Y T L T I S S L Q P E D V A T Y Y C Q Q Y SI L P W T F G G G T K V E I K R; b) [SEQ ID NO: 16]D I Q M T Q S P S S L S A S V G D R V T I T CQ A S Q G I N N Y L N W Y Q Q K P G K A P K LL I H Y T S G L H A G V P S R F S G S G S G TD Y T L T I S S L E P E D V A T Y Y C Q Q Y SI L P W T F G G G T K V E I K R; or c) [SEQ ID NO: 17]D I Q M T Q S P S S L S A S V G D R V T I T CQ A S Q G I N N Y L N W Y Q Q K P G K A P K LL I H Y T S G L H A G V P S R F S G S G S G TD F T L T I S S L E P E D V A T Y Y C Q Q Y SI L P W T F G G G T K V E I K R.

For example, the antibody or antigen-binding fragment thereof maycomprise a light chain variable region having an amino acid sequence ofany one of SEQ ID NOs: 15 to 17.

In one embodiment, the antibody or antigen-binding fragment thereofcomprises a murine heavy chain variable region which comprises orconsists of the amino acid sequence of any one of SEQ ID NO: 1 and amurine light chain variable region which comprises or consists of theamino acid sequence of any one of SEQ ID NO: 2.

Alternatively, the antibody or antigen-binding fragment thereof maycomprise a humanised heavy chain variable region which comprises orconsists of the amino acid sequence of any one of SEQ ID NOs: 8 to 11and a humanised light chain variable region which comprises or consistsof the amino acid sequence of any one of SEQ ID NOs: 15 to 17.

For example, the antibody or antigen-binding fragment thereof maycomprise:

-   -   a) a heavy chain variable region which comprises or consists of        the amino acid sequence of SEQ ID NO: 8 and a light chain        variable region which comprises or consists of the amino acid        sequence of SEQ ID NO: 15;    -   b) a heavy chain variable region which comprises or consists of        the amino acid sequence of SEQ ID NO: 9 and a light chain        variable region which comprises or consists of the amino acid        sequence of SEQ ID NO: 15;    -   c) a heavy chain variable region which comprises or consists of        the amino acid sequence of SEQ ID NO: 10 and a light chain        variable region which comprises or consists of the amino acid        sequence of SEQ ID NO: 15;    -   d) a heavy chain variable region which comprises or consists of        the amino acid sequence of SEQ ID NO: 11 and a light chain        variable region which comprises or consists of the amino acid        sequence of SEQ ID NO: 15;    -   e) a heavy chain variable region which comprises or consists of        the amino acid sequence of SEQ ID NO: 8 and a light chain        variable region which comprises or consists of the amino acid        sequence of SEQ ID NO: 16;    -   f) a heavy chain variable region which comprises or consists of        the amino acid sequence of SEQ ID NO: 9 and a light chain        variable region which comprises or consists of the amino acid        sequence of SEQ ID NO: 16;    -   g) a heavy chain variable region which comprises or consists of        the amino acid sequence of SEQ ID NO: 10 and a light chain        variable region which comprises or consists of the amino acid        sequence of SEQ ID NO: 16;    -   h) a heavy chain variable region which comprises or consists of        the amino acid sequence of SEQ ID NO: 11 and a light chain        variable region which comprises or consists of the amino acid        sequence of SEQ ID NO: 16;    -   i) a heavy chain variable region which comprises or consists of        the amino acid sequence of SEQ ID NO: 8 and a light chain        variable region which comprises or consists of the amino acid        sequence of SEQ ID NO: 17;    -   j) a heavy chain variable region which comprises or consists of        the amino acid sequence of SEQ ID NO: 9 and a light chain        variable region which comprises or consists of the amino acid        sequence of SEQ ID NO: 17;    -   k) a heavy chain variable region which comprises or consists of        the amino acid sequence of SEQ ID NO: 10 and a light chain        variable region which comprises or consists of the amino acid        sequence of SEQ ID NO: 17; or    -   l) a heavy chain variable region which comprises or consists of        the amino acid sequence of SEQ ID NO: 11 and a light chain        variable region which comprises or consists of the amino acid        sequence of SEQ ID NO: 17.

It will be appreciated by persons skilled in the art that theabove-defined humanised antibodies or antigen-binding fragments of theinvention may further comprise a heavy chain constant region, or partthereof (see below).

In one embodiment, the antibody polypeptide comprises a CH1, CH2 and/orCH3 region of an IgG heavy chain (such as an IgG1, IgG2, IgG3 or IgG4heavy chain). Thus, the antibody polypeptide may comprise part or all ofthe constant regions from an IgG1 heavy chain. For example, the antibodypolypeptide may be a Fab fragment comprising CH1 and CL constantregions, combined with any of the above-defined heavy and light variableregions respectively.

Likewise, the above-defined antibodies or antigen-binding fragments ofthe invention may further comprise a light chain constant region, orpart thereof (see below). For example, the antibody polypeptide maycomprise a CL region from a kappa or lambda light chain.

For example, the antibody polypeptide may comprise the followingconstant regions:

(a) Ig kappa chain C region (Homo sapiens)(UnitProt Accession No. P01834) [SEQ ID NO: 18]TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQWKVDNALQSGN SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC(b) Ig gamma-1 chain C region (Homo sapiens)(UnitProt Accession No. P01857) [SEQ ID NO: 19]ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVSWNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQTYICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGGPSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNWYVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGKEYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREEMTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPVLDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK

In an alternative embodiment, naturally occurring variants of the aboveconstant regions may be utilised (e.g. see Jefferis & Lefranc, 2009,MAbs 1(4):332-8, the disclosures of which are incorporated herein byreference). For example, the light china constant region may comprise orconsist of SEQ ID NO: 18 having a W40R and/or V83L mutation and/or theheavy china constant region may comprise or consist of SEQ ID NO: 19having a K97R, D239E and/or L241M mutation, or without the C-terminallysine/K (wherein the position of the amino acid mutations is definedusing the Eu Numbering Scheme, which differs from the numbering in SEQID NOS: 18 and 19; see Edelman et al., 1969, Proc. Natl. Acad. Sci. USA,63:78-85, the disclosures of which are incorporated herein byreference).

Thus, exemplary antibody polypeptides of the invention comprise:

-   -   (a) a heavy chain comprising a variable region of SEQ ID NO: 1,        8, 9, 10 or 11 together with a constant region of SEQ ID NO: 19;        and    -   (b) a light chain comprising a variable region of SEQ ID NO: 2,        15, 16 or 17 together with a constant region of SEQ ID NO: 18.

In a related embodiment, the antibody polypeptide may comprise anantibody Fc-region (e.g. the CH2 and CH3 regions of an IgG heavy chain).It will be appreciated by a skilled person that the Fc portion may befrom an IgG antibody, or from a different class of antibody (such asIgM, IgA, IgD or IgE). In one embodiment, the Fc region is from an IgG1,IgG2, IgG3 or IgG4 antibody.

The Fc region may be naturally-occurring (e.g. part of an endogenouslyproduced antibody) or may be artificial (e.g. comprising one or morepoint mutations relative to a naturally-occurring Fc region).

As is well documented in the art, the Fc region of an antibody mediatesits serum half-life and effector functions, such as complement-dependentcytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) andantibody-dependent cell phagocytosis (ADCP).

Engineering the Fc region of a therapeutic monoclonal antibody or Fcfusion protein allows the generation of molecules that are better suitedto the pharmacology activity required of them (Strohl, 2009, Curr OpinBiotechnol 20(6):685-91, the disclosures of which are incorporatedherein by reference).

(a) Engineered Fc Regions for Increased Half-life

One approach to improve the efficacy of a therapeutic antibody is toincrease its serum persistence, thereby allowing higher circulatinglevels, less frequent administration and reduced doses.

The half-life of an IgG depends on its pH-dependent binding to theneonatal receptor FcRn. FcRn, which is expressed on the surface ofendothelial cells, binds the IgG in a pH-dependent manner and protectsit from degradation.

Some antibodies that selectively bind the FcRn at pH 6.0, but not pH7.4, exhibit a higher half-life in a variety of animal models.

Several mutations located at the interface between the CH2 and CH3domains, such as T250Q/M428L (Hinton et al., 2004, J Biol Chem.279(8):6213-6, the disclosures of which are incorporated herein byreference) and M252Y/S254T/T256E+H433K/N434F (Vaccaro et al., 2005, Nat.Biotechnol. 23(10):1283-8, the disclosures of which are incorporatedherein by reference), have been shown to increase the binding affinityto FcRn and the half-life of IgG1 in vivo.

(b) Engineered Fc Regions for Altered Effector Function

Depending on the therapeutic antibody or Fc fusion protein application,it may be desired to either reduce or increase the effector function(such as ADCC).

For antibodies that target cell-surface molecules, especially those onimmune cells, abrogating effector functions may be required for certainclinical indications.

Conversely, for antibodies intended for oncology use (such as in thetreatment of leukemias and solid tumours; see below), increasingeffector functions may improve the therapeutic activity.

The four human IgG isotypes bind the activating Fcγ receptors (FcγRI,FcγRIIa, FcγRIIIa), the inhibitory FcγRIIb receptor, and the firstcomponent of complement (C1q) with different affinities, yielding verydifferent effector functions (Bruhns et al., 2009, Blood.113(16):3716-25, the disclosures of which are incorporated herein byreference).

Binding of IgG to the FcγRs or C1q depends on residues located in thehinge region and the CH2 domain. Two regions of the CH2 domain arecritical for FcγRs and C1q binding, and have unique sequences in IgG2and IgG4. Substitutions into human IgG1 of IgG2 residues at positions233-236 and IgG4 residues at positions 327, 330 and 331 were shown togreatly reduce ADCC and CDC (Armour et al., 1999, Eur J Immunol.29(8):2613-24; Shields et al., 2001, J Biol Chem. 276(9):6591-604, thedisclosures of which are incorporated herein by reference). Furthermore,Idusogie et al. demonstrated that alanine substitution at differentpositions, including K322, significantly reduced complement activation(Idusogie et al., 2000, J Immunol. 164(8):4178-84, the disclosures ofwhich are incorporated herein by reference). Similarly, mutations in theCH2 domain of murine IgG2A were shown to reduce the binding to FcγRI,and C1q (Steurer. et al., 1995. J Immunol. 155(3):1165-74, thedisclosures of which are incorporated herein by reference).

Numerous mutations have been made in the CH2 domain of human IgG1 andtheir effect on ADCC and CDC tested in vitro (see references citedabove). Notably, alanine substitution at position 333 was reported toincrease both ADCC and CDC (Shields et al., 2001, supra; Steurer et al.,1995, supra). Lazar et al. described a triple mutant (S239D/I332E/A330L)with a higher affinity for FcγRIIIa and a lower affinity for FcγRIIbresulting in enhanced ADCC (Lazar et al., 2006, PNAS 103(11):4005-4010,the disclosures of which are incorporated herein by reference). The samemutations were used to generate an antibody with increased ADCC (Ryan etal., 2007, Mol. Cancer Ther. 6:3009-3018, the disclosures of which areincorporated herein by reference). Richards et al. studied a slightlydifferent triple mutant (S239D/I332E/G236A) with improved FcγRIIIaaffinity and FcγRIIa/FcγRIIb ratio that mediates enhanced phagocytosisof target cells by macrophages (Richards et al., 2008. Mol Cancer Ther.7(8):2517-27, the disclosures of which are incorporated herein byreference).

Due to their lack of effector functions, IgG4 antibodies represent apreferred IgG subclass for receptor blocking without cell depletion(i.e. inhibition of IL-1 signalling). IgG4 molecules can exchangehalf-molecules in a dynamic process termed Fab-arm exchange. Thisphenomenon can also occur in vivo between therapeutic antibodies andendogenous IgG4.

The S228P mutation has been shown to prevent this recombination processallowing the design of less unpredictable therapeutic IgG4 antibodies(Labrijn et al., 2009, Nat Biotechnol. 27(8):767-71, the disclosures ofwhich are incorporated herein by reference).

Examples of engineered Fc regions are shown in Table 1 and Example Jbelow.

TABLE 1 Examples of Engineered Fc Effector Isotype Species Mutations*FcR/C1q Binding Function IgG1 Human T250Q/M428L¹ Increased bindingIncreased half- to FcRn life IgG1 Human M252Y/S254T/T256E + Increasedbinding Increased half- H433K/N434F² to FcRn life IgG1 HumanM428L/N434S³ Increased binding Increased half- to FcRn life IgG1 HumanE233P/L234V/L235A/?G236 + Reduced binding Reduced ADCCA327G/A330S/P331S^(4,5) to FcγRI and CDC IgG1 Human S239D/S298A/I332E +Increased binding Increased ADCC S239D/A330L/I332E⁶ to FcγRIIIa IgG1Human S239D/I332E⁷ Increased binding Increased ADCC to FcγRIIIa IgG1Human S298D/E333A/K334A⁸ Increased binding Increased ADCC to FcγRIIIaIgG1 Human E333A⁹ Increased binding Increased ADCC to FcγRIIIa and CDCIgG1 Human P257I/Q311¹⁰ Increased binding Unchanged half- to FcRn lifeIgG1 Human K326W/E333S¹¹ Increased binding Increased CDC to C1q IgG1Human S239D/I332E/G236A¹² Increased Increased FcγRIIa/FcγRIIb macrophageratio phagocytosis IgG1 Human K322A⁸ Reduced binding Reduced DCC to C1qN297S Reduced (abrogated) ADCC N297Q Reduced (abrogated) ADCC R292P +V305I +/− F243L¹³ Increased ADCC P247I/A339Q¹⁴ Increased ADCC IgG4 HumanS228P¹⁵ — Reduced Fab- arm exchange IgG2a Mouse L235E +E318A/K320A/K322A¹¹ Reduced binding Reduced ADCC to FcγRI and C1q andCDC *The position of the Fc amino acid mutations is defined using the EuNumbering Scheme, which differs from the numbering in SEQ ID NOS: 18 and19 above; see Edelman et al., 1969, Proc. Natl. Acad. Sci. USA, 63:78-85) References to Table 1 ¹Hinton et al 2004 J. Biol. Chem.279(8):6213-6) ²Vaccaro et al. 2005 Nat Biotechnol. 23(10): 1283-8)³Zalevsky et al 2010 Nat. Biotechnology 28(2):157-159 ⁴Armour K L. etal., 1999. Eur J Immunol. 29(8):2613-24 ⁵Shields RL. et al., 2001. JBiol Chem. 276(9):6591-604 ⁶Masuda et al. 2007, Mol Immunol.44(12):3122-31 ⁷Bushfield et al 2014, Leukemia 28(11):2213-21 ⁸Okazakiet al. 2004, J Mol Biol. ;336(5):1239-49 ⁹Idusogie et al., 2000, JImmunol. 164(8):4178-84 ¹⁰Datta-Mannan A. et al., 2007, Drug Metab.Dispos. 35:86-94 ¹¹Steurer W. et al., 1995, J Immunol. 155(3):1165-74¹²Richards et al. 2008 Mol Cancer There. 7(8):2517-27 ¹³U.S. Pat. No.7,960,512 B2 ¹⁴EP 2 213 683 ¹⁵Labrijn AF. et al., 2009, Nat Biotechnol.27(8):767-71

In a further embodiment, the effector function of the Fc region may bealtered through modification of the carbohydrate moieties within the CH2domain therein.

For example, it is known that therapeutic antibodies lacking or low infucose residues in the Fc region may exhibit enhanced ADCC activity inhumans (for example, see Peipp et al., 2008, Blood 112(6):2390-9,Yamane-Ohnuki & Satoh, 2009, MAbs 1(3):230-26, lida et al., 2009, BMCCancer 9;58 (the disclosures of which are incorporated herein byreference). Low fucose antibody polypeptides may be produced byexpression in cells cultured in a medium containing an inhibitor ofmannosidase, such as kinfunensine (see Example I below).

Other methods to modify glycosylation of an antibody into a low fucoseformat include the use of the bacterial enzymeGDP-6-deoxy-D-lyxo-4-hexulose reductase in cells not able to metaboliserhamnose (e.g. using the GlymaxX® technology of ProBioGen AG, Berlin,Germany).

Another method to create low fucose antibodies is by inhibition ordepletion of alpha-(1,6)-fucosyltransferase in the antibody-producingcells (e.g. using the Potelligent® CHOK1SV technology of Lonza Ltd,Basel, Switzerland).

As noted above, the antibody polypeptides of the invention may exert aninhibitory action on IL-1 signalling (see Example E), either in additionto or in the absence of any Fc-mediated effector functions.

In one embodiment, the antibody polypeptides of the invention may exertan inhibitory action on one or more additional (or alternative)cytokines within the IL-1 superfamily, including but not limited toIL-33 and/or IL-36.

Interleukin-33 (IL-33) induces helper T cells, mast cells, eosinophilsand basophils to produce type 2 cytokines. This cytokine was previouslynamed NF-HEV ‘nuclear factor (NF) in high endothelial venules’ (HEVs)since it was originally identified in these specialized cells. IL-33mediates its biological effects by interacting with the receptors ST2(also known as IL1RL1) and IL-1 Receptor Accessory Protein (IL1RAP),activating intracellular molecules in the NF-κB and MAP kinase signalingpathways that drive production of type 2 cytokines (e.g. IL-5 and IL-13)from polarised Th2 cells. The induction of type 2 cytokines by IL-33 invivo is believed to induce the severe pathological changes observed inmucosal organs following administration of IL-33.

Interleukin-36 (IL-36) is a cytokine that predominantly acts on naiveCD4+ T cells via the IL-36 receptor. It is known to activate NF-κB andmitogen-activated protein kinases to play a role in skin pathology. Ithas also been found to activate T cell proliferation and release ofIL-2.

It will be appreciated by persons skilled in the art that the antibodypolypeptide of the invention may inhibit IL-1, IL-33 and/or IL-36signalling in whole or in part. For example, signalling may be inhibitedby at least 10%, 20%, 30%, 50%, 75% or more relative to signalling inthe absence of the polypeptide of the invention.

The degree of inhibition of IL-1, IL-33 and/or IL-36 signalling by thepolypeptide of the invention may be determined using methods well knownin the art.

For example, inhibition of IL-1 signalling may be measured as describedin Example E below.

Likewise, inhibition of IL-33 signalling may be measured as described inExample E.

Inhibition of IL-36 signalling may be measured by methods known in theart. For example, IL-36 stimulation of synovial fibroblasts leads toNF-κB and MAP kinase activation. Alternatively, IL-36-α, -β and -γincrease T-cell proliferation in response to antiCD3/anti-CD28stimulation (see Vigne et al., 2012, Blood 120(17):3478-87, thedisclosures of which are incorporated herein by reference).

In one embodiment, the antibody or antigen-binding fragment thereof mayfurther comprise a moiety for increasing the in vivo half-life of theantibody or antigen-binding fragment, such as but not limited topolyethylene glycol (PEG), human serum albumin, glycosylation groups,fatty acids and dextran. Such further moieties may be conjugated orotherwise combined with the binding moiety using methods well known inthe art.

Any one or more of the following known methods of improving thehalf-life of proteins may be used for this purpose:

(a) PEGylation

A widely used method for improving the half-life of proteins is thecovalent linking of polyethylene glycol (PEG) moieties to the protein.PEGs are water-soluble polymers that due to their large hydrodynamicvolume create a shield around the pegylated drug [Molineux, G.,Pegylation: engineering improved pharmaceuticals for enhanced therapy.Cancer Treat Rev, 2002. 28 Suppl A: p. 13-6, the disclosures of whichare incorporated herein by reference]. Pegylated proteins exhibit adecreased renal clearance and proteolysis, reduced toxicity, reducedimmunogenicity and an increased solubility [Veronese, F. M. and J. M.Harris, Introduction and overview of peptide and protein pegylation. AdvDrug Deliv Rev, 2002. 54(4): p. 453-6, Chapman, A. P., PEGylatedantibodies and antibody fragments for improved therapy: a review. AdvDrug Deliv Rev, 2002. 54(4): p. 531-45.]. Pegylation has been employedfor several protein-based drugs including the first pegylated moleculesasparaginase and adenosine deaminase [Veronese, F. M. and J. M. Harris,Introduction and overview of peptide and protein pegylation. Adv DrugDeliv Rev, 2002. 54(4): p. 453-6, Veronese, F. M. and G. Pasut,PEGylation, successful approach to drug delivery. Drug Discov Today,2005. 10(21): p. 1451-8, the disclosures of which are incorporatedherein by reference].

In order to obtain a successfully pegylated protein, with a maximallyincreased half-life and retained biological activity, several parametersthat may affect the outcome are of importance and should be taken intoconsideration. The PEG molecules may differ, and PEG variants that havebeen used for pegylation of proteins include PEG and monomethoxy-PEG. Inaddition, they can be either linear or branched [Wang, Y. S., et al.,Structural and biological characterization of pegylated recombinantinterferon alpha-2b and its therapeutic implications. Adv Drug DelivRev, 2002. 54(4): p. 547-70]. The size of the PEG molecules used mayvary and PEG moieties ranging in size between 1 and 40 kDa have beenlinked to proteins [Wang, Y. S., et al., Structural and biologicalcharacterization of pegylated recombinant interferon alpha-2b and itstherapeutic implications. Adv Drug Deliv Rev, 2002. 54(4): p. 547-70,Sato, H., Enzymatic procedure for site-specific pegylation of proteins.Adv Drug Deliv Rev, 2002. 54(4): p. 487-504, Bowen, S., et al.,Relationship between molecular mass and duration of activity ofpolyethylene glycol conjugated granulocyte colony-stimulating factormutein. Exp Hematol, 1999. 27(3): p. 425-32, Chapman, A. P., et al.,Therapeutic antibody fragments with prolonged in vivo half-lives. NatBiotechnol, 1999. 17(8): p. 780-3]. In addition, the number of PEGmoieties attached to the protein may vary, and examples of between oneand six PEG units being attached to proteins have been reported [Wang,Y. S., et al., Structural and biological characterization of pegylatedrecombinant interferon alpha-2b and its therapeutic implications. AdvDrug Deliv Rev, 2002. 54(4): p. 547-70, Bowen, S., et al., Relationshipbetween molecular mass and duration of activity of polyethylene glycolconjugated granulocyte colony-stimulating factor mutein. Exp Hematol,1999. 27(3): p. 425-32]. Furthermore, the presence or absence of alinker between PEG as well as various reactive groups for conjugationhave been utilised. Thus, PEG may be linked to N-terminal amino groups,or to amino acid residues with reactive amino or hydroxyl groups (Lys,His, Ser, Thr and Tyr) directly or by using γ-amino butyric acid as alinker. In addition, PEG may be coupled to carboxyl (Asp, Glu,C-terminal) or sulfhydryl (Cys) groups. Finally, Gin residues may bespecifically pegylated using the enzyme transglutaminase and alkylaminederivatives of PEG has been described [Sato, H., Enzymatic procedure forsite-specific pegylation of proteins. Adv Drug Deliv Rev, 2002. 54(4):p. 487-504].

It has been shown that increasing the extent of pegylation results in anincreased in vivo half-life. However, it will be appreciated by personsskilled in the art that the pegylation process will need to be optimisedfor a particular antibody polypeptide on an individual basis.

PEG may be coupled at naturally occurring disulphide bonds as describedin WO 2005/007197, the disclosures of which are incorporated herein byreference. Disulfide bonds can be stabilised through the addition of achemical bridge which does not compromise the tertiary structure of theprotein. This allows the conjugating thiol selectivity of the twosulphurs comprising a disulfide bond to be utilised to create a bridgefor the site-specific attachment of PEG. Thereby, the need to engineerresidues into a peptide for attachment of to target molecules iscircumvented.

A variety of alternative block copolymers may also be covalentlyconjugated as described in WO 2003/059973, the disclosures of which areincorporated herein by reference. Therapeutic polymeric conjugates canexhibit improved thermal properties, crystallisation, adhesion,swelling, coating, pH dependent conformation and biodistribution.Furthermore, they can achieve prolonged circulation, release of thebioactive in the proteolytic and acidic environment of the secondarylysosome after cellular uptake of the conjugate by pinocytosis and morefavourable physicochemical properties due to the characteristics oflarge molecules (e.g. increased drug solubility in biological fluids).Co-block copolymers, comprising hydrophilic and hydrophobic blocks, formpolymeric micelles in solution. Upon micelle disassociation, theindividual block copolymer molecules are safely excreted.

(b) Fusion Proteins

Where the invention comprises or otherwise resides in the use of anantibody mimic (see above), the following types of fusion protein may beuseful to extend half-life in vivo.

IgG Fusion Proteins

Human immunoglobulin G (IgG) molecules have circulating half-lives ofapproximately 20 days. The Fc portion of IgG molecules have beenextensively used for the creation of fusion proteins consisting of an Fcpart and a protein with a therapeutic use. Such fusion proteins exhibita prolonged half-life compared to their Fc-lacking counterparts. Forexample, this strategy was used for the development of etanercept, ananti-rheumatic drug composed of a fusion protein between the solublehuman p75 tumour necrosis factor receptor and the Fc portion of humanIgG [Goldenberg, M. M., Etanercept, a novel drug for the treatment ofpatients with severe, active rheumatoid arthritis. Clin Ther, 1999.21(1): p. 75-87; discussion 1-2, the disclosures of which areincorporated herein by reference].

Fc-linked proteins are produced by creating fusion proteins between Fcand the antigen-binding region of the polypeptide of interest bystandard genetic engineering protocols. The Fc group is fused to theC-terminus of the protein of interest. Due to the presence of cysteineresidues in the hinge region of IgG, Fc fusion proteins are expressed asdisulfide-linked homodimers. This further increases their effective sizeand circulating half-lives. In addition, homodimeric constructs may havean increased functional activity due to improved avidity for itsreceptor/ligand compared to the corresponding monomeric form.

Human Serum Albumin Fusion Proteins

Human serum albumin (HSA) is the most abundant naturally occurring bloodprotein in the circulation and has a half-life of 19 days [Osborn, B.L., et al., Pharmacokinetic and pharmacodynamic studies of a human serumalbumin-interferon-alpha fusion protein in cynomolgus monkeys. JPharmacol Exp Ther, 2002. 303(2): p. 540-8, the disclosures of which areincorporated herein by reference]. Thus, HSA is a suitable fusionpartner for the creation of fusion proteins with improved half-life. HSAfusion proteins exhibit a prolonged half-life due to the capability ofHSA to stabilize the protein towards proteolysis and increasing theresidence time in the body [Veronese, F. M. and J. M. Harris,Introduction and overview of peptide and protein pegylation. Adv DrugDeliv Rev, 2002. 54(4): p. 453-6]. HSA fusion proteins, including IL-2,IFN-α and -β and growth hormone (GH), have been produced and shown tohave improved pharmacokinetic properties. Albuferon (HSA-IFN-α) andalbutropin (HSA-GH) exhibit half-lives that are 18 and 6 times longer incynomolgus monkeys, respectively, than the respective counterpartslacking an HSA group [Osborn, B. L., et al., Pharmacokinetic andpharmacodynamic studies of a human serum albumin-interferon-alpha fusionprotein in cynomolgus monkeys. J Pharmacol Exp Ther, 2002. 303(2): p.540-8, Osborn, B. L., et al., Albutropin: a growth hormone-albuminfusion with improved pharmacokinetics and pharmacodynamics in rats andmonkeys. Eur J Pharmacol, 2002. 456(1-3): p. 149-58].

HSA-linked proteins are produced by creating fusion proteins between HSAand the protein of interest by standard genetic engineering protocols.The HSA group may be added at either the N- or the C-terminus. Since themodification is added to the terminus of the protein, the risk ofinterfering with the structure of the protein and thus with its functionis considerably less compared to modifications such as pegylation in theinterior of the protein. In addition, the chance of avoidinginterference with the active site of the protein is increased by thefact that the HSA group may be added at either the N- or C-terminus ofthe protein of interest [Osborn, B. L., et al., Pharmacokinetic andpharmacodynamic studies of a human serum albumin-interferon-alpha fusionprotein in cynomolgus monkeys. J Pharmacol Exp Ther, 2002. 303(2): p.540-8, Osborn, B. L., et al., Albutropin: a growth hormone-albuminfusion with improved pharmacokinetics and pharmacodynamics in rats andmonkeys. Eur J Pharmacol, 2002. 456(1-3): p. 149-58, Syed, S., K. E.Kelly, and W. P. Sheffield, Inhibition of thrombin by hirudingenetically fused to wild-type or mutant antithrombin. Thromb Res, 1996.84(6): p. 419-29], depending on which is more likely to result in afusion protein with maintained biological activity. Thus, in the case ofalbuferon and albutropin, the C-terminus of the HSA was fused with theN-terminus of IFN-α or GH, respectively, creation of a functionallyactive hirudin-HSA fusion protein, the HSA group had to be fused to theC-terminus of hirudin. These results indicate that the properties of thetarget protein determine whether fusion at the N- or C-terminus isoptimal.

(c) Glycosylation

The introduction of new sialic acid-containing carbohydrates into aprotein (glycoengineering) has been shown to improve in vivo half-life.This method may be used for naturally glycosylated proteins or forproteins that normally lack glycosylation [Elliott, S., et al.,Enhancement of therapeutic protein in vivo activities throughglycoengineering. Nat Biotechnol, 2003. 21(4): p. 414-21, thedisclosures of which are incorporated herein by reference].

Glycosylation of proteins may be in the form of N-linked or O-linkedcarbohydrates. N-linked carbohydrates are typically attached toconsensus sequences (Asn-X-Ser/Thr) where X is any amino acid exceptproline. O-glycosylation occurs at Ser/Thr residues.

For the production of glycosylated proteins, the introduction of novelglycosylation sites may be required. For glycosylation to occur,expression may be performed in yeast, insect or mammalian cell systems.However, the glycosylation pattern in yeast cells is different thanmammalian cells, generating hyper-glycosylated proteins, associated witha risk of increased immunogenicity. In contrast, insect cells may bepreferred since the glycosylation pattern is similar to that inmammalian cells whereas cell cycles are shorter and therefore expressionprocess faster. Darbepoetin-α is an example of a modified humanerythropoietin expressed in CHO cells. It contains two extraN-glycosylation sites, resulting in a three times improved in vivohalf-life [Elliott, S., et al., Enhancement of therapeutic protein invivo activities through glycoengineering. Nat Biotechnol, 2003. 21(4):p. 414-21].

An alternative method of glycosylation is the chemical addition ofcarbohydrate groups to proteins. In this method, the protein isexpressed naked, e.g. in E. coli. Following expression and purification,the protein is glycosylated in a fully synthetic cell-free process. Themethod offers great flexibility in terms of number, size and type ofcarbohydrate to be added.

(d) Fatty Acid Acylation/Myristoylation

Fatty acids have a high affinity and high capacity of HSA binding. Thischaracteristic can be utilized for improving the half-life of proteins.Thus, fatty acyl can be attached to amino acids of proteins, thusgenerating fatty acyl acylated proteins. Upon reaching the circulation,the fatty acyl group is capable of binding to circulating HSA, resultingin an improved in vivo half-life of the protein.

This method was used for the development of Insulin detemir, which wasfatty acyl acylated with myristate at Lys^(B29) by treatment of insulinwith fatty acid hydroxyl-succinimide esters in dimethyl formamide/DMSO[Kurtzhals, P., et al., Albumin binding of insulins acylated with fattyacids: characterization of the ligand-protein interaction andcorrelation between binding affinity and timing of the insulin effect invivo. Biochem J, 1995. 312 (Pt 3): p. 725-31, Hamilton-Wessler, M., etal., Mechanism of protracted metabolic effects of fatty acid acylatedinsulin, NN304, in dogs: retention of NN304 by albumin. Diabetologia,1999. 42(10): p. 1254-63, the disclosures of which are incorporatedherein by reference]. This generated an insulin analogue with increasedin vivo half-life due to binding of HSA.

(e) Dextran

Dextran results in an immobilization of the protein, resulting in a slowrelease and thereby improves the half-life of the protein.Dextran-streptokinase, has been marketed in Russia for thrombolytictherapy. In addition, insulin, somatostatin (which is used for therapyand diagnosis of tumours expressing somatostatin receptors) and theribosome-inactivating drug trichosantin conjugated to dextran, had asignificantly improved half-lives [Baudys, M., et al., Extending insulinaction in vivo by conjugation to carboxymethyl dextran. Bioconjug Chem,1998. 9(2): p. 176-83, Chan, W. L., et al., Lowering of trichosanthinimmunogenicity by site-specific coupling to dextran. Biochem Pharmacol,1999. 57(8): p. 927-34, Wulbrand, U., et al., A novel somatostatinconjugate with a high affinity to all five somatostatin receptorsubtypes. Cancer, 2002. 94(4 Suppl): p. 1293-7, the disclosures of whichare incorporated herein by reference].

In addition to protein-based pharmaceuticals, dextran has been used forimproving the half-life of antibiotics and cytotoxic drugs [Yura, H., etal., Synthesis and pharmacokinetics of a novel macromolecular prodrug ofTacrolimus (FK506), FK506-dextran conjugate. J Control Release, 1999.57(1): p. 87-99, Nakashima, M., et al., In vitro characteristics and invivo plasma disposition of cisplatin conjugated with oxidized anddicarboxymethylated dextrans. Biol Pharm Bull, 1999. 22(7): p. 756-61,Kim, D. S., Y. J. Jung, and Y. M. Kim, Synthesis and properties ofdextran-linked ampicillin. Drug Dev Ind Pharm, 2001. 27(1): p. 97-101,the disclosures of which are incorporated herein by reference].

Dextran conjugation is carried out by reductive amination usingperiodate-activated dextran or by the use of cyanogens bromide[Wulbrand, U., et al., A novel somatostatin conjugate with a highaffinity to all five somatostatin receptor subtypes. Cancer, 2002. 94(4Suppl): p. 1293-7, Kim, D. S., Y. J. Jung, and Y. M. Kim, Synthesis andproperties of dextran-linked ampicillin. Drug Dev Ind Pharm, 2001.27(1): p. 97-101, the disclosures of which are incorporated herein byreference]. The dextran used may vary in size, and dextran ranging from9 to 82 kDa have been used [Kim, D. S., Y. J. Jung, and Y. M. Kim,Synthesis and properties of dextran-linked ampicillin. Drug Dev IndPharm, 2001. 27(1): p. 97-101, Behe, M., et al., Biodistribution, bloodhalf-life, and receptor binding of a somatostatin-dextran conjugate. MedOncol, 2001. 18(1): p. 59-64, the disclosures of which are incorporatedherein by reference].

In addition to improving the half-life of drugs, dextran conjugation mayalso reduce immunogenicity [Chan, W. L., et al., Lowering oftrichosanthin immunogenicity by site-specific coupling to dextran.Biochem Pharmacol, 1999. 57(8): p. 927-34, the disclosures of which areincorporated herein by reference].

Thus, in one embodiment of the first aspect of the invention, thepolypeptide of the invention is or comprises a “fusion” polypeptide.

In addition to being fused to a moiety in order to improvepharmacokinetic properties, it will be appreciated that the polypeptideof the invention may also be fused to a polypeptide such asglutathione-S-transferase (GST) or protein A in order to facilitatepurification of said polypeptide. Examples of such fusions are wellknown to those skilled in the art. Similarly, the said polypeptide maybe fused to an oligo-histidine tag, such as His6, or to an epitoperecognised by an antibody such as the well-known Myc tag epitope.Fusions to any variant or derivative of said polypeptide are alsoincluded in the scope of the invention. It will be appreciated thatfusions (or variants, derivatives or fusions thereof) which retain orimprove desirable properties, such as IL-1R binding properties or invivo half-life are preferred.

Thus, the fusion may comprise an amino acid sequence as detailed abovetogether with a further portion which confers a desirable feature on thesaid polypeptide of the invention; for example, the portion may usefulin detecting or isolating the polypeptide, or promoting cellular uptakeof the polypeptide. The portion may be, for example, a biotin moiety, aradioactive moiety, a fluorescent moiety, for example a smallfluorophore or a green fluorescent protein (GFP) fluorophore, as wellknown to those skilled in the art. The moiety may be an immunogenic tag,for example a Myc tag, as known to those skilled in the art or may be alipophilic molecule or polypeptide domain that is capable of promotingcellular uptake of the polypeptide, as known to those skilled in theart.

It will be appreciated by persons skilled in the art that the antibodypolypeptides of the invention may comprise or consist of one or moreamino acids which have been modified or derivatised.

Chemical derivatives of one or more amino acids may be achieved byreaction with a functional side group. Such derivatised moleculesinclude, for example, those molecules in which free amino groups havebeen derivatised to form amine hydrochlorides, p-toluene sulphonylgroups, carboxybenzoxy groups, t-butyloxycarbonyl groups, chloroacetylgroups or formyl groups. Free carboxyl groups may be derivatised to formsalts, methyl and ethyl esters or other types of esters and hydrazides.Free hydroxyl groups may be derivatised to form O-acyl or O-alkylderivatives. Also included as chemical derivatives are those peptideswhich contain naturally occurring amino acid derivatives of the twentystandard amino acids. For example: 4-hydroxyproline may be substitutedfor proline; 5-hydroxylysine may be substituted for lysine;3-methylhistidine may be substituted for histidine; homoserine may besubstituted for serine and ornithine for lysine. Derivatives alsoinclude peptides containing one or more additions or deletions as longas the requisite activity is maintained. Other included modificationsare amidation, amino terminal acylation (e.g. acetylation orthioglycolic acid amidation), terminal carboxylamidation (e.g. withammonia or methylamine), and the like terminal modifications.

It will be further appreciated by persons skilled in the art thatpeptidomimetic compounds may also be useful. The term ‘peptidomimetic’refers to a compound that mimics the conformation and desirable featuresof a particular peptide as a therapeutic agent.

For example, the said polypeptide includes not only molecules in whichamino acid residues are joined by peptide (—CO—NH—) linkages but alsomolecules in which the peptide bond is reversed. Such retro-inversopeptidomimetics may be made using methods known in the art, for examplesuch as those described in Meziere et al. (1997) J. Immunol. 159,3230-3237, which is incorporated herein by reference. This approachinvolves making pseudo-peptides containing changes involving thebackbone, and not the orientation of side chains. Retro-inversepeptides, which contain NH—CO bonds instead of CO—NH peptide bonds, aremuch more resistant to proteolysis. Alternatively, the said polypeptidemay be a peptidomimetic compound wherein one or more of the amino acidresidues are linked by a -y(CH₂NH)— bond in place of the conventionalamide linkage.

In a further alternative, the peptide bond may be dispensed withaltogether provided that an appropriate linker moiety which retains thespacing between the carbon atoms of the amino acid residues is used; itmay be advantageous for the linker moiety to have substantially the samecharge distribution and substantially the same planarity as a peptidebond.

It will also be appreciated that the said polypeptide may convenientlybe blocked at its N- or C-terminus so as to help reduce susceptibilityto exo-proteolytic digestion.

A variety of un-coded or modified amino acids such as D-amino acids andN-methyl amino acids have also been used to modify mammalian peptides.In addition, a presumed bioactive conformation may be stabilised by acovalent modification, such as cyclisation or by incorporation of lactamor other types of bridges, for example see Veber et al., 1978, Proc.Natl. Acad. Sci. USA 75:2636 and Thursell et al., 1983, Biochem.Biophys. Res. Comm. 111:166, which are incorporated herein by reference.

The antibody polypeptides of the invention may be augmented with afunctional moiety to facilitate their intended use, for example as adiagnostic (e.g. in vivo imaging) agent or therapeutic agent. Thus, inone embodiment, the antibody polypeptide is linked, directly orindirectly, to a therapeutic moiety.

In one embodiment, the antibody or antigen-binding fragment thereofaccording to any one of the preceding claim further comprising atherapeutic (e.g. cytotoxic) moiety.

Any suitable therapeutic moiety may be used. A suitable therapeuticmoiety is one that is capable of reducing or inhibiting the growth, orin particular killing, a cancer cell (or associated stem cells orprogenitor cells). For example, the therapeutic agent may be a cytotoxicmoiety. The cytotoxic moiety may comprise or consist of one or moreradioisotopes. For example, the one or more radioisotopes may each beindependently selected from the group consisting of beta-emitters,Auger-emitters, conversion electron-emitters, alpha-emitters, and lowphoton energy-emitters. It may be desired that the one or moreradioisotopes each independently has an emission pattern of locallyabsorbed energy that creates a high absorbed dose in the vicinity of theagent. Exemplary radioisotopes may include long-range beta-emitters,such as ⁹⁰Y, ³²P, ¹⁸⁶Re/¹⁸⁸Re; ¹⁶⁶Ho, ⁷⁶As/⁷⁷As, ⁸⁹Sr, ¹⁵³Sm; mediumrange beta-emitters, such as ¹³¹I, ¹⁷⁷Lu, ⁶⁷Cu, ¹⁶¹Tb, ¹⁰⁵Rh; low-energybeta-emitters, such as ⁴⁵Ca or ³⁵S; conversion or Auger-emitters, suchas ⁵¹Cr, ⁶⁷Ga, ⁹⁹Tc^(m), ¹¹¹In, ^(114m)In, ¹²³I, ¹²⁵I, ²⁰¹Tl; andalpha-emitters, such as ²¹²Bi, ²¹³Bi, ²²³Ac, ²²⁵Ac, ²¹²Pb, ²⁵⁵Fm, ²²³Ra,¹⁴⁹Tb and ²²¹At. Other radionuclides are available and will be possibleto use for therapy.

In one preferred embodiment, the antibody polypeptide is linked to (orotherwise labelled with) the radioisotope ¹⁷⁷Lu.

Alternatively, the therapeutic moiety may comprise or consist of one ormore therapeutic (such as cytotoxic) drugs, for example, a cytostaticdrug; an anti-androgen drug; cortisone and derivatives thereof; aphosphonate; a testosterone-5-α-reductase inhibitor; a boron addend; acytokine; thapsigargin and its metabolites; a toxin (such as saporin orcalicheamicin); a chemotherapeutic agent (such as an antimetabolite); orany other therapeutic or cytotoxic drug useful in the treatment ofcancers.

Exemplary therapeutic/cytotoxic drugs may, for example, include:

-   -   Cytostatics, in particular those with dose-limiting        side-effects, including but not limited to cyclophosamide,        chlorambucil, ifosfamide, busulphane, lomustine, taxanes,        estramustine phosphate and other nitrogen mustards, antibiotics        (including doxorubicine, calicheamicines and esperamicine),        vinca alkaloids, azaridines, platinum-containing compounds,        endostatin, alkyl sulfonates, nitrosoureas, triazenes, folic        acid analoges, pyrimidine analoges, purine analogs, enzymes,        substituted urea, methyl-hydrazine derivatives, daunorubicin,        amphipathic amines,    -   Anti-androgens such as flutamide and bicalutamide and        metabolites thereof;    -   Cortisone and derivatives thereof;    -   Phosphonates such as diphophonate and buphosphonate;    -   Testosterone-5-α-reductase inhibitors;    -   Boron addends;    -   Cytokines;    -   Thapsigargin and its metabolites.

Alternatively, the cytotoxic moiety may comprise or consist of one ormore moieties suitable for use in activation therapy, such as photonactivation therapy, neutron activation therapy, neutron-induced Augerelectron therapy, synchrotron irradiation therapy or low energy X-rayphoton activation therapy.

For example, with the antibody polypeptides of the invention there willbe the potential of using synchrotron radiation (or low energy X-rays)for the advancement of radiotherapy, primarily focusing on so calledphoto-activation radiotherapy (PAT), in which the local energydeposition from external X-ray irradiation is enhanced in the cancertissue by the interaction with a pre-administered, high-Ztumour-targeting agent.

The PAT treatment modality utilises monochromatic X-rays from asynchrotron source, such as provided by the ID17 biomedical beamline atthe European Synchrotron Radiation Facility (ESRF) in Grenoble, and asanticipated to be available at other facilities in the future such asthe new Swedish synchrotron facility, Max-IV.

Research on “induced Auger electron tumour therapy”, to be conducted atthe coming European Spallation Source (ESS) in Lund, provides a furtherpotential treatment modality. Reactor-produced thermal and semi-thermalneutrons have for long been used for Boron-Neutron-Capture-Therapy,BNCT, both for pre-clinical experiments and for treatment of braintumours with the induced alpha-particles and the recoil nucleus (⁷L)that give a high locally absorbed energy. A similar approach is to useneutrons and suitable tumour-targeting molecules labelled with stablenuclei with high cross-section for neutrons. Antibodies or peptides canfor instance be labelled with stable Gadolinium (¹⁵⁷Gd) and act as thetarget molecule for the neutrons that are captured by the Gd-nucleus, socalled Gadolinium Neutron Capture Therapy (GdNCT). By Monte Carlotechniques, the dose distribution in the tumour and the surroundingtissues is calculated as it results from γ-photons, neutrons, nuclearrecoils, as well as characteristic x-rays, internal conversion andAuger-electrons from gadolinium or other potential elements.

Optionally, the antibody polypeptide of the invention may furthercomprise a detectable moiety. For example, a detectable moiety maycomprise or consist of a radioisotope, such as a radioisotope selectedfrom the group consisting of ^(99m)Tc, ¹¹¹In, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ⁸⁹Zr,¹²³I and ²⁰¹Tl Optionally, the agent may comprise a pair of detectableand cytotoxic radionuclides, such as ⁸⁶Y/⁹⁰Y or ¹²⁴I/²¹¹At.Alternatively, the antibody polypeptide may comprise a radioisotope thatis capable of simultaneously acting in a multi-modal manner as adetectable moiety and also as a cytotoxic moiety to provide so-called“Multimodality theragnostics”. The binding moieties may thus be coupledto nanoparticles that have the capability of multi-imaging (for example,SPECT, PET, MRI, Optical, or Ultrasound) together with therapeuticcapability using cytotoxic drugs, such as radionuclides or chemotherapyagents.

Alternatively, the detectable moiety may comprise or consist of aparamagnetic isotope, such as a paramagnetic isotope is selected fromthe group consisting of ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr and ⁵⁶Fe.

In the case that the antibody polypeptide comprises a detectable moiety,then the detectable moiety may be detectable by an imaging techniquesuch as SPECT, PET, MRI, optical or ultrasound imaging.

Therapeutic and/or detectable moieties (such as a radioisotope,cytotoxic moiety or the like) may be linked directly, or indirectly, tothe antibody or fragment thereof. Suitable linkers are known in the artand include, for example, prosthetic groups, non-phenolic linkers(derivatives of N-succimidyl-benzoates; dodecaborate), chelatingmoieties of both macrocyclics and acyclic chelators, such as derivativesof 1,4,7,10-tetraazacyclododecane-1,4,7,10,tetraacetic acid (DOTA),deferoxamine (DFO), derivatives of diethylenetriaminepentaacetic avid(DTPA), derivatives ofS-2-(4-Isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triaceticacid (NOTA) and derivatives of1,4,8,11-tetraazacyclodocedan-1,4,8,11-tetraacetic acid (TETA),derivatives of3,6,9,15-Tetraazabicyclo[9.3.1]-pentadeca-1(15),11,13-triene-4-(S)-(4-isothiocyanato-benzyl)-3,6,9-triaceticacid (PCTA), derivatives of5-S-(4-Aminobenzyl)-1-oxa-4,7,10-triazacyclododecane-4,7,10-tris(aceticacid) (DO3A) and other chelating moieties.

One preferred linker is DTPA, for example as used in¹⁷⁷Lu-DTPA-[antibody polypeptide of the invention]. A further preferredlinker is deferoxamine, DFO, for example as used in ⁸⁹Zr-DFO-[antibodypolypeptide of the invention].

However, it will be appreciated by persons skilled in the art that somemedical uses of the antibody polypeptides of the invention will notrequire the presence of a cytotoxic or diagnostic moiety.

Thus, where the therapeutic effect of the antibody of the invention ismediated by inhibition of IL-1 signalling (or IL-33 and/or IL-36signalling), a ‘naked’ antibody polypeptide may be suitable. Forexample, where the therapeutic effect is mediated by a direct effect ofthe antibody of the invention on immune cells, e.g. to reduceinflammation, it may be advantageous for the antibody to lack anycytotoxic activity.

As discussed above, methods for the production of antibody polypeptidesof the invention are well known in the art.

Conveniently, the antibody polypeptide is or comprises a recombinantpolypeptide. Suitable methods for the production of such recombinantpolypeptides are well known in the art, such as expression inprokaryotic or eukaryotic hosts cells (for example, see Green &Sambrook, 2012, Molecular Cloning, A Laboratory Manual, Fourth Edition,Cold Spring Harbor, N.Y., the relevant disclosures in which document arehereby incorporated by reference).

Antibody polypeptides of the invention can also be produced using acommercially available in vitro translation system, such as rabbitreticulocyte lysate or wheatgerm lysate (available from Promega).Preferably, the translation system is rabbit reticulocyte lysate.Conveniently, the translation system may be coupled to a transcriptionsystem, such as the TNT transcription-translation system (Promega). Thissystem has the advantage of producing suitable mRNA transcript from anencoding DNA polynucleotide in the same reaction as the translation.

It will be appreciated by persons skilled in the art that antibodypolypeptides of the invention may alternatively be synthesisedartificially, for example using well known liquid-phase or solid phasesynthesis techniques (such as t-Boc or Fmoc solid-phase peptidesynthesis).

A second aspect of the invention provides an isolated nucleic acidmolecule encoding an antibody or antigen-binding fragment of the firstaspect of the invention, or a component polypeptide chain thereof. By“nucleic acid molecule” we include DNA (e.g. genomic DNA orcomplementary DNA) and mRNA molecules, which may be single- ordouble-stranded. By “isolated” we mean that the nucleic acid molecule isnot located or otherwise provided within a cell.

In one embodiment, the nucleic acid molecule is a cDNA molecule.

It will be appreciated by persons skilled in the art that the nucleicacid molecule may be codon-optimised for expression of the antibodypolypeptide in a particular host cell, e.g. for expression in humancells (for example, see Angov, 2011, Biotechnol. J. 6(6):650-659, thedisclosures of which are incorporated herein by reference).

Also included within the scope of the invention are the following:

-   (a) a third aspect of the invention provides a vector (such as an    expression vector) comprising a nucleic acid molecule according to    the second aspect of the invention;-   (b) a fourth aspect of the invention provides a host cell (such as a    mammalian cell, e.g. human cell, or Chinese hamster ovary cell, e.g.    CHOK1SV cells) comprising a nucleic acid molecule according to the    second aspect of the invention or a vector according to the third    aspect of the invention; and-   (c) a fifth aspect of the invention provides a method of making an    antibody polypeptide according to the first aspect of the invention    comprising culturing a population of host cells according to the    fourth aspect of the invention under conditions in which said    polypeptide is expressed, and isolating the polypeptide therefrom.

A sixth aspect of the invention provides a pharmaceutical compositioncomprising a pharmaceutically effective amount of an antibody orantigen-binding fragment according to the first aspect of the inventionand a pharmaceutically-acceptable diluent, carrier, adjuvant orexcipient.

It will be appreciated by persons skilled in the art that additionalcompounds may also be included in the pharmaceutical compositions,including, chelating agents such as EDTA, citrate, EGTA or glutathione.

The pharmaceutical compositions may be prepared in a manner known in theart that is sufficiently storage stable and suitable for administrationto humans and animals. For example, the pharmaceutical compositions maybe lyophilised, e.g. through freeze drying, spray drying, spray cooling,or through use of particle formation from supercritical particleformation.

By “pharmaceutically acceptable” we mean a non-toxic material that doesnot decrease the effectiveness of the IL1RAP-binding activity of theantibody polypeptide of the invention. Such pharmaceutically acceptablebuffers, carriers or excipients are well-known in the art (seeRemington's Pharmaceutical Sciences, 18th edition, A. R Gennaro, Ed.,Mack Publishing Company (1990) and handbook of PharmaceuticalExcipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press (2000), thedisclosures of which are incorporated herein by reference).

The term “buffer” is intended to mean an aqueous solution containing anacid-base mixture with the purpose of stabilising pH. Examples ofbuffers are Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes,HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate,borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO, BES, CABS, cacodylate,CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole,imidazolelactic acid, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO andTES.

The term “diluent” is intended to mean an aqueous or non-aqueoussolution with the purpose of diluting the antibody polypeptide in thepharmaceutical preparation. The diluent may be one or more of saline,water, polyethylene glycol, propylene glycol, ethanol or oils (such assafflower oil, corn oil, peanut oil, cottonseed oil or sesame oil).

The term “adjuvant” is intended to mean any compound added to theformulation to increase the biological effect of the antibodypolypeptide of the invention. The adjuvant may be one or more of zinc,copper or silver salts with different anions, for example, but notlimited to fluoride, chloride, bromide, iodide, thiocyanate, sulfite,hydroxide, phosphate, carbonate, lactate, glycolate, citrate, borate,tartrate, and acetates of different acyl composition. The adjuvant mayalso be cationic polymers such as cationic cellulose ethers, cationiccellulose esters, deacetylated hyaluronic acid, chitosan, cationicdendrimers, cationic synthetic polymers such as poly(vinyl imidazole),and cationic polypeptides such as polyhistidine, polylysine,polyarginine, and peptides containing these amino acids.

The excipient may be one or more of carbohydrates, polymers, lipids andminerals. Examples of carbohydrates include lactose, glucose, sucrose,mannitol, and cyclodextrines, which are added to the composition, e.g.for facilitating lyophilisation. Examples of polymers are starch,cellulose ethers, cellulose carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose,alginates, carageenans, hyaluronic acid and derivatives thereof,polyacrylic acid, polysulphonate, polyethylenglycol/polyethylene oxide,polyethyleneoxide/polypropylene oxide copolymers,polyvinylalcohol/polyvinylacetate of different degree of hydrolysis, andpolyvinylpyrrolidone, all of different molecular weight, which are addedto the composition, e.g., for viscosity control, for achievingbioadhesion, or for protecting the lipid from chemical and proteolyticdegradation. Examples of lipids are fatty acids, phospholipids, mono-,di-, and triglycerides, ceramides, sphingolipids and glycolipids, all ofdifferent acyl chain length and saturation, egg lecithin, soy lecithin,hydrogenated egg and soy lecithin, which are added to the compositionfor reasons similar to those for polymers. Examples of minerals aretalc, magnesium oxide, zinc oxide and titanium oxide, which are added tothe composition to obtain benefits such as reduction of liquidaccumulation or advantageous pigment properties.

The antibody polypeptides of the invention may be formulated into anytype of pharmaceutical composition known in the art to be suitable forthe delivery thereof.

In one embodiment, the pharmaceutical compositions of the invention maybe in the form of a liposome, in which the antibody polypeptide iscombined, in addition to other pharmaceutically acceptable carriers,with amphipathic agents such as lipids, which exist in aggregated formsas micelles, insoluble monolayers and liquid crystals. Suitable lipidsfor liposomal formulation include, without limitation, monoglycerides,diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bileacids, and the like. Suitable lipids also include the lipids abovemodified by poly(ethylene glycol) in the polar headgroup for prolongingbloodstream circulation time. Preparation of such liposomal formulationsis can be found in for example U.S. Pat. No. 4,235,871, the disclosuresof which are incorporated herein by reference.

The pharmaceutical compositions of the invention may also be in the formof biodegradable microspheres. Aliphatic polyesters, such as poly(lacticacid) (PLA), poly(glycolic acid) (PGA), copolymers of PLA and PGA (PLGA)or poly(caprolactone) (PCL), and polyanhydrides have been widely used asbiodegradable polymers in the production of microspheres. Preparationsof such microspheres can be found in U.S. Pat. No. 5,851,451 and in EP 0213 303, the disclosures of which are incorporated herein by reference.

In a further embodiment, the pharmaceutical compositions of theinvention are provided in the form of polymer gels, where polymers suchas starch, cellulose ethers, cellulose carboxymethylcellulose,hydroxypropylmethyl cellulose, hydroxyethyl cellulose, ethylhydroxyethylcellulose, alginates, carageenans, hyaluronic acid and derivativesthereof, polyacrylic acid, polyvinyl imidazole, polysulphonate,polyethylenglycol/polyethylene oxide, polyethyleneoxide/polypropyleneoxide copolymers, polyvinylalcohol/polyvinylacetate of different degreeof hydrolysis, and polyvinylpyrrolidone are used for thickening of thesolution containing the agent. The polymers may also comprise gelatin orcollagen.

Alternatively, the antibody polypeptide may simply be dissolved insaline, water, polyethylene glycol, propylene glycol, ethanol or oils(such as safflower oil, corn oil, peanut oil, cottonseed oil or sesameoil), tragacanth gum, and/or various buffers.

It will be appreciated that the pharmaceutical compositions of theinvention may include ions and a defined pH for potentiation of actionof the active antibody polypeptide. Additionally, the compositions maybe subjected to conventional pharmaceutical operations such assterilisation and/or may contain conventional adjuvants such aspreservatives, stabilisers, wetting agents, emulsifiers, buffers,fillers, etc.

The pharmaceutical compositions according to the invention may beadministered via any suitable route known to those skilled in the art.Thus, possible routes of administration include parenteral (intravenous,subcutaneous, and intramuscular), topical, ocular, nasal, pulmonar,buccal, oral, parenteral, vaginal and rectal. Also administration fromimplants is possible.

In one preferred embodiment, the pharmaceutical compositions areadministered parenterally, for example, intravenously,intracerebroventricularly, intraarticularly, intra-arterially,intraperitoneally, intrathecally, intraventricularly, intrasternally,intracranially, intramuscularly or subcutaneously, or they may beadministered by infusion techniques. They are conveniently used in theform of a sterile aqueous solution which may contain other substances,for example, enough salts or glucose to make the solution isotonic withblood. The aqueous solutions should be suitably buffered (preferably toa pH of from 3 to 9), if necessary. The preparation of suitableparenteral formulations under sterile conditions is readily accomplishedby standard pharmaceutical techniques well known to those skilled in theart.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example sealed ampoules and vials, and may be stored ina freeze-dried (lyophilised) condition requiring only the addition ofthe sterile liquid carrier, for example water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

Thus, the pharmaceutical compositions of the invention are particularlysuitable for parenteral, e.g. intravenous, administration.

Alternatively, the pharmaceutical compositions may be administeredintranasally or by inhalation (for example, in the form of an aerosolspray presentation from a pressurised container, pump, spray ornebuliser with the use of a suitable propellant, such asdichlorodifluoromethane, trichlorofluoro-methane,dichlorotetrafluoro-ethane, a hydrofluoroalkane such as1,1,1,2-tetrafluoroethane (HFA 134A3 or 1,1,1,2,3,3,3-heptafluoropropane(HFA 227EA3), carbon dioxide or other suitable gas). In the case of apressurised aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. The pressurised container, pump,spray or nebuliser may contain a solution or suspension of the activepolypeptide, e.g. using a mixture of ethanol and the propellant as thesolvent, which may additionally contain a lubricant, e.g. sorbitantrioleate. Capsules and cartridges (made, for example, from gelatin) foruse in an inhaler or insufflator may be formulated to contain a powdermix of a compound of the invention and a suitable powder base such aslactose or starch.

Aerosol or dry powder formulations are preferably arranged so that eachmetered dose or ‘puff’ contains at least 1 mg of a compound of theinvention for delivery to the patient. It will be appreciated that theoverall daily dose with an aerosol will vary from patient to patient,and may be administered in a single dose or, more usually, in divideddoses throughout the day.

Alternatively, the antibody polypeptides of the invention can beadministered in the form of a suppository or pessary, or they may beapplied topically in the form of a lotion, solution, cream, ointment ordusting powder. The compounds of the invention may also be transdermallyadministered, for example, by the use of a skin patch. They may also beadministered by the ocular route.

For ophthalmic use, the antibody polypeptides of the invention can beformulated as micronised suspensions in isotonic, pH adjusted, sterilesaline, or, preferably, as solutions in isotonic, pH adjusted, sterilesaline, optionally in combination with a preservative such as abenzylalkonium chloride. Alternatively, they may be formulated in anointment such as petrolatum.

For application topically to the skin, the antibody polypeptide of theinvention can be formulated as a suitable ointment containing the activecompound suspended or dissolved in, for example, a mixture with one ormore of the following: mineral oil, liquid petrolatum, white petrolatum,propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifyingwax and water. Alternatively, they can be formulated as a suitablelotion or cream, suspended or dissolved in, for example, a mixture ofone or more of the following: mineral oil, sorbitan monostearate, apolyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax,cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

The pharmaceutical compositions will be administered to a patient in apharmaceutically effective dose. A ‘therapeutically effective amount’,or ‘effective amount’, or ‘therapeutically effective’, as used herein,refers to that amount which provides a therapeutic effect for a givencondition and administration regimen. This is a predetermined quantityof active material calculated to produce a desired therapeutic effect inassociation with the required additive and diluent, i.e. a carrier oradministration vehicle. Further, it is intended to mean an amountsufficient to reduce and most preferably prevent, a clinicallysignificant deficit in the activity, function and response of the host.Alternatively, a therapeutically effective amount is sufficient to causean improvement in a clinically significant condition in a host. As isappreciated by those skilled in the art, the amount of a compound mayvary depending on its specific activity.

Suitable dosage amounts may contain a predetermined quantity of activecomposition calculated to produce the desired therapeutic effect inassociation with the required diluent. In the methods and use formanufacture of compositions of the invention, a therapeuticallyeffective amount of the active component is provided. A therapeuticallyeffective amount can be determined by the ordinary skilled medical orveterinary worker based on patient characteristics, such as age, weight,sex, condition, complications, other diseases, etc., as is well known inthe art. The administration of the pharmaceutically effective dose canbe carried out both by single administration in the form of anindividual dose unit or else several smaller dose units and also bymultiple administrations of subdivided doses at specific intervals.Alternatively, the does may be provided as a continuous infusion over aprolonged period.

In the context of diagnostic use of the antibody polypeptides of theinvention, a ‘pharmaceutically effective amount’, or ‘effective amount’,or ‘diagnostically effective’, as used herein, refers to that amountwhich provides a detectable signal for diagnosis, e.g. for in vivoimaging purposes.

The antibody polypeptides can be formulated at various concentrations,depending on the efficacy/toxicity of the polypeptide being used. Forexample, the formulation may comprise the active antibody polypeptide ata concentration of between 0.1 μM and 1 mM, more preferably between 1 μMand 500 μM, between 500 μM and 1 mM, between 300 μM and 700 μM, between1 μM and 100 μM, between 100 μM and 200 μM, between 200 μM and 300 μM,between 300 μM and 400 μM, between 400 μM and 500 μM, between 500 μM and600 μM, between 600 μM and 700 μM, between 800 μM and 900 μM or between900 μM and 1 mM. Typically, the formulation comprises the activeantibody polypeptide at a concentration of between 300 μM and 700 μM.

Typically, the therapeutic dose of the antibody polypeptide (with orwithout a therapeutic moiety) in a human patient will be in the range of100 μg to 1 g per administration (based on a body weight of 70 kg, e.g.between 300 μg to 700 mg per administration). For example, the maximumtherapeutic dose may be in the range of 0.1 to 10 mg/kg peradministration, e.g. between 0.1 and 5 mg/kg or between 1 and 5 mg/kg orbetween 0.1 and 2 mg/kg. It will be appreciated that such a dose may beadministered at different intervals, as determined by theoncologist/physician; for example, a dose may be administered daily,twice-weekly, weekly, bi-weekly or monthly.

It will be appreciated by persons skilled in the art that thepharmaceutical compositions of the invention may be administered aloneor in combination with other therapeutic agents used in the treatment ofcancers, such as antimetabolites, alkylating agents, anthracyclines andother cytotoxic antibiotics, vinca alkyloids, etoposide, platinumcompounds, taxanes, topoisomerase I inhibitors, antiproliferativeimmunosuppressants, corticosteroids, sex hormones and hormoneantagonists, and other therapeutic antibodies (such as trastuzumab).

It will be further appreciated by persons skilled in the art that thepolypeptides and pharmaceutical formulations of the present inventionhave utility in both the medical and veterinary fields. Thus, themethods of the invention may be used in the treatment of both human andnon-human animals (such as horses, dogs and cats). Preferably, however,the patient is human.

For veterinary use, a compound of the invention is administered as asuitably acceptable formulation in accordance with normal veterinarypractice and the veterinary surgeon will determine the dosing regimenand route of administration which will be most appropriate for aparticular animal.

A seventh aspect of the invention provides an antibody orantigen-binding fragment thereof according to the first aspect of theinvention for use in medicine.

In one embodiment, the antibody polypeptides and formulations of theinvention may be used to treat patients or subjects who suffer from orare at risk of suffering from a disease or indication for which IL1RAPis a biomarker.

Thus, a related eighth aspect of the invention provides an antibody orantigen-binding fragment thereof according to the first aspect of theinvention for use in inducing cell death and/or inhibiting the growthand/or proliferation of pathological cells associated with a neoplasticdisorder in a subject, or stem cells or progenitor cells thereof,wherein the cells express IL1RAP.

A further related ninth aspect of the invention provides an antibody orantigen-binding fragment according to the first aspect of the inventionfor use in the treatment and/or diagnosis of a neoplastic disorder in asubject, wherein the neoplastic disorder is associated with cellsexpressing IL1RAP.

By ‘treatment’ we include both therapeutic and prophylactic treatment ofthe patient. The term ‘prophylactic’ is used to encompass the use of anagent, or formulation thereof, as described herein which either preventsor reduces the likelihood of a neoplastic disorder, or the spread,dissemination, or metastasis of cancer cells in a patient or subject.The term ‘prophylactic’ also encompasses the use of an agent, orformulation thereof, as described herein to prevent recurrence of aneoplastic disorder in a patient who has previously been treated for theneoplastic disorder.

By “diagnosis” we include the detection of cancerous cells, either invivo (i.e. within the body of a patient) or ex vivo (i.e. within atissue or cell sample removed from the body of a patient).

By “a neoplastic disorder associated with cells expressing IL1RAP” weinclude such disorders wherein the pathological cells which areresponsible, directly or indirectly, for the disorder express IL1RAP onthe cell surface. It will be appreciated that the cells expressingIL1RAP may be cancer cells, e.g. tumour cells, per se. In addition, suchcells include pathological stem cells (i.e. cancer stem cells, or CSCs)and progenitor cells which are responsible, directly or indirectly, forthe development of a neoplastic disorder in an individual. Examples ofCSCs are disclosed in Visvader & Lindeman, 2008, Nat Rev Cancer8:755-768, the disclosures of which are incorporated herein byreference.

Alternatively, or in addition, the cells expressing IL1RAP may beassociated indirectly with the neoplastic disorder, for example, theymay mediate cellular processes required for the neoplastic cells tosurvive. The antibody agent of the invention may in this event targetcells essential for the blood supply of the tumour (angiogenesis) orcells inhibiting a beneficial immune response directed against themalignant cells (e.g. suppressive macrophages or T-cells).

Depending upon whether it is therapeutically desirable to kill thetarget cells expressing IL1RAP, an antibody or antigen-binding fragmentaccording to the first aspect of the invention may be used that itcapable of inducing ADCC. For example, where the target cells IL1RAP arecancer cells (such as CML, AML, ALL, melanoma, lung cancer cells, etc)it may be advantageous for the antibody or antigen-binding fragment tobe capable of inducing ADCC in order to eliminate such cells. However,it will be appreciated that a therapeutic benefit may also be achievedusing an antibody or antigen-binding fragment that lacks ADCC activity,for example through inhibition of IL-1 (or IL-33 or IL-36) signallingleading to reduced angiogenesis in the vicinity of a tumour.

In one embodiment, the neoplastic disorder is a neoplastic hematologicdisorder.

For example, the antibody or antigen-binding fragment thereof may be foruse in the treatment and/or diagnosis of a neoplastic disorder selectedfrom the group consisting of chronic myeloid leukemia (CML),myeloproliferative disorders (MPD), myelodysplastic syndrome (MDS),acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML).

In a further embodiment, the antibody or antigen-binding fragmentthereof is for use in the treatment and/or diagnosis of a neoplasticdisorder associated with the formation of solid tumours within thesubject's body.

Thus, the antibody or antigen-binding fragment thereof may be for use inthe treatment of a neoplastic disorder selected from the groupconsisting of prostate cancer, breast cancer, lung cancer, colorectalcancer, melanomas, bladder cancer, brain/CNS cancer, cervical cancer,oesophageal cancer, gastric cancer, head/neck cancer, kidney cancer,liver cancer, lymphomas, ovarian cancer, pancreatic cancer, andsarcomas.

In relation to the therapeutic and prophylactic aspects of theinvention, it will be appreciated by persons skilled in the art thatbinding of the antibody polypeptide to IL1RAP present on the surface ofthe cells associated with the neoplastic disorder may lead to amodulation (i.e. an increase or decrease) of a biological activity ofIL1RAP. However, such a modulatory effect is not essential; for example,the antibody polypeptides of the invention may elicit a therapeutic andprophylactic effect simply by virtue of binding to IL1RAP on the surfaceof the cells associated with the solid tumour, which in turn may triggerthe immune system to induce cell death (e.g. by ADCC and/or by thepresence within the agent of a cytotoxic/radioactive moiety).

By “biological activity of IL1RAP” we include any interaction orsignalling event which involves IL1RAP on the cells associated with theneoplastic disorder. For example, in one embodiment the antibodypolypeptide is capable of blocking binding of one or more co-receptorsto IL1RAP (such as IL1R1, ST2, C-KIT and/or IL1RL2).

Such inhibition of the biological activity of IL1RAP by an antibodypolypeptide of the invention may be in whole or in part. For example,the agent may inhibit the biological activity of IL1RAP by at least 10%,preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, and mostpreferably by 100% compared to the biological activity of IL1RAP incells associated with the neoplastic disorder which have not beenexposed to the antibody polypeptide. In a preferred embodiment, theantibody polypeptide is capable of inhibiting the biological activity ofIL1RAP by 50% or more compared to the biological activity of IL1RAP incells associated with the neoplastic disorder which have not beenexposed to the antibody polypeptide.

Likewise, it will be appreciated that inhibition of growth and/orproliferation of the cells associated with the neoplastic disorder maybe in whole or in part. For example, the antibody polypeptide mayinhibit the growth and/or proliferation of the cells associated with theneoplastic disorder by at least 10%, preferably at least 20%, 30%, 40%,50%, 60%, 70%, 80% or 90%, and most preferably by 100% compared to thegrowth and/or proliferation of cells associated with the neoplasticdisorder which have not been exposed to the antibody polypeptide.

A tenth aspect of the invention provides an of an antibody orantigen-binding fragment thereof according to the first aspect of theinvention in the preparation of a medicament for the treatment ordiagnosis of a neoplastic disorder in a subject, wherein the neoplasticdisorder is associated with cells expressing IL1RAP.

In one embodiment, the neoplastic disorder is a neoplastic hematologicdisorder.

For example, the antibody or antigen-binding fragment thereof may be foruse in the treatment and/or diagnosis of a neoplastic disorder selectedfrom the group consisting of chronic myeloid leukemia (CML),myeloproliferative disorders (MPD), myelodysplastic syndrome (MDS),acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML).

In a further embodiment, the antibody or antigen-binding fragmentthereof is for use in the treatment and/or diagnosis of a neoplasticdisorder associated with the formation of solid tumours within thesubject's body.

Thus, the antibody or antigen-binding fragment thereof may be for use inthe treatment of a neoplastic disorder selected from the groupconsisting of prostate cancer, breast cancer, lung cancer, colorectalcancer, melanomas, bladder cancer, brain/CNS cancer, cervical cancer,oesophageal cancer, gastric cancer, head/neck cancer, kidney cancer,liver cancer, lymphomas, ovarian cancer, pancreatic cancer, andsarcomas.

An eleventh aspect of the invention provides a method for the treatmentor diagnosis of a neoplastic disorder in a subject, comprising the stepof administering to the subject an effective amount of an antibody orantigen-binding fragment thereof according to the first aspect of theinvention, wherein the neoplastic disorder is associated with cellsexpressing IL1RAP.

In one embodiment, the neoplastic disorder is a neoplastic hematologicdisorder.

For example, the method may be for use in the treatment and/or diagnosisof a neoplastic disorder selected from the group consisting of chronicmyeloid leukemia (CML), myeloproliferative disorders (MPD),myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL) andacute myeloid leukemia (AML).

In a further embodiment, the method is for use in the treatment and/ordiagnosis of a neoplastic disorder associated with the formation ofsolid tumours within the subject's body.

Thus, the method may be for use in the treatment of a neoplasticdisorder selected from the group consisting of prostate cancer, breastcancer, lung cancer, colorectal cancer, melanomas, bladder cancer,brain/CNS cancer, cervical cancer, oesophageal cancer, gastric cancer,head/neck cancer, kidney cancer, liver cancer, lymphomas, ovariancancer, pancreatic cancer, and sarcomas.

In a further embodiment, the antibody polypeptides and formulations ofthe invention may be used to treat patients or subjects who suffer fromor are at risk of suffering a disease or condition susceptible totreatment with an inhibitor of IL-1 (or IL-33 or IL-36) signalling.

Thus, a twelfth aspect of the invention provides an antibody orantigen-binding fragment thereof according to the first aspect of theinvention for use in the treatment of a disease or condition susceptibleto treatment with an inhibitor of IL-1 signalling.

Such conditions or disease states are well known in the art (seeDinarello et al., 2012, Nature Reviews 11:633-652 and Dinarello, 2014,Mol. Med. 20(suppl. 1):S43-S58, the disclosures of which areincorporated herein by reference) and include, but are not limited to,the following:

-   -   Rheumatoid arthritis, all types of juvenile arthritis including        systemic onset juvenile idiopathic arthritis (SOJIA),        osteoarthritis, familial cold auto-inflammatory syndrome (FCAS),        Muckle-Wells disease, neonatal onset multi-system inflammatory        disease (NOMID), familial Mediterranean fever (FMF), pyogenic        arthritis pyoderma gangrenosum and acne (PAPA) syndrome, adult        onset Still's disease, hyper IgD syndrome, type 2 diabetes        mellitus, macrophage activation syndrome, TNF        receptor-associated periodic syndrome, Blau disease, ankylosing        spondylitis, Sweets disease, lupus arthritis, Alzheimer's        disease, psoriasis, asthma, atherosclerosis, sarcoidosis, atopic        dermatitis, systemic lupus erythematosus, bullous pemphigoid,        type I diabetes mellitus, chronic obstructive pulmonary disease,        Helicobacter pylori gastritis, inflammatory bowel disease        (including ulcerative colitis and Crohn's disease), Hepatitis C,        ischaemia-reperfusion injury, multiple sclerosis, Neisserial or        pneumococcal meningitis, tuberculosis, Bechet's syndrome, septic        shock, graft versus host disease, asthma, type I diabetes,        Alzheimer's disease, atherosclerosis, adult T cell leukaemia,        multiple myeloma, periodontitis, obesity and obesity-related        diseases (for example, metabolic syndrome, cardiomegaly,        congestive heart failure, varicose veins, polycystic ovarian        syndrome, gastroesophageal reflux disease (GERD), fatty liver        disease, colorectal cancer, breast cancer, uterine cancer,        chronic renal failure, stroke and hyperuricemia), intervertebral        disc disease, irritable bowel syndrome, Schnitzler syndrome,        allergy/atopic dermatitis and gout.

Blockade of IL-1 signalling is also believed to be beneficial in thetreatment of myocardial infarction. An extensive clinical trial iscurrently seeking to confirm the efficacy of IL1B antibody blockade(using Canakinumab) following myocardial infarction (the CANTOS trail;see Ridker et al., 2011, Am Heart Journal 162(4):597-605, thedisclosures of which are incorporated herein by reference).

For such indications, it will be appreciated that a therapeutic benefitmay also be achieved using an antibody or antigen-binding fragment thatbinds IL1RAP and thereby blocking IL-1 (or IL-33 or IL-36) signallingassociated with immune cells. Such antibody could be modified to lackADCC activity.

A thirteenth aspect of the invention provides the use of an antibody orantigen-binding fragment thereof according to the first aspect of theinvention in the preparation of a medicament for the treatment of adisease or condition susceptible to treatment with an inhibitor of IL-1(or IL_33 or IL-36) signalling.

A fourteenth aspect of the invention provides a method for the treatmentof a disease or condition susceptible to treatment with an inhibitor ofIL-1 (or IL_33 or IL-36) signalling in a subject, comprising the step ofadministering to the subject an effective amount of an antibody orantigen-binding fragment thereof according to the first aspect of theinvention.

A fifteenth aspect of the invention provides a method for theADCC-mediated treatment or augmentation of a disease or conditionsusceptible to treatment with an inhibitor of IL-1 (or IL-33 or IL-36)signalling in a subject, comprising the step of administering to thesubject an effective amount of an antibody or antigen-binding fragmentthereof according to the first aspect of the invention capable ofinducing ADCC.

A sixteenth aspect of the invention provides an in vitro method for thedetection of cancer cells in a subject, the method comprising:

-   -   (a) providing a sample of cells (e.g. white blood        stem/progenitor cells or biopsy tissue) from a subject to be        tested;    -   (b) optionally, extracting and/or purifying the cells present in        the sample;    -   (c) contacting an antibody or antigen-binding fragment thereof        according to the first aspect of the invention with cells        present in the sample;    -   (d) determining whether the antibody polypeptide binds to the        cells        wherein the binding of the antibody polypeptide to the cells is        indicative of the presence of cancer cells in the tissue of a        subject.

A seventeenth aspect of the invention provides an in vitro method foridentifying a patient with cancer who would benefit from treatment withan antibody or antigen-binding fragment thereof according to the firstaspect of the invention, the method comprising:

-   -   (a) providing a sample of cancer cells (e.g. white blood        stem/progenitor cells or biopsy tissue) from a patient to be        tested;    -   (b) optionally, extracting and/or purifying the cells present in        the sample;    -   (c) contacting an antibody or antigen-binding fragment thereof        according to the first aspect of the invention with cells        present in the sample;    -   (d) determining whether the antibody polypeptide binds to the        cells        wherein the binding of the antibody polypeptide to the cancer        cells is indicative of a patient who would benefit from        treatment with an antibody or antigen-binding fragment thereof        according to the first aspect of the invention.

Persons skilled in the art will appreciate that there are many ways toperform such an assay. For example, the immunoassay could be eitherhomogeneous or, more preferably, heterogeneous. The assay could also beperformed in either a competitive or, more preferably, a non-competitiveformat.

In one embodiment, IL1RAP expression on blood samples (leukemia) orbiopsies (solid tumours) from patients is measured using flow cytometryor immunohistochemistry, with expression above a threshold value beingindicative of a patient who would benefit from treatment with anantibody or antigen-binding fragment thereof according to the firstaspect of the invention.

In preferred embodiments of the above in vitro methods, step (d) isperformed by flow cytometry, immunohistochemistry or ELISA.

However, other assays suitable for detecting antibody-antigeninteractions in vitro may be used.

A eighteenth aspect of the invention provides a method for treating apatient with cancer, the method comprising administering to a subjectidentified as having cancer using a method according to the sixteenth orseventeenth aspects of the invention a therapeutic agent effective inthe treatment of said cancer. In one embodiment, the example therapeuticagent is an antibody polypeptide according to the first aspect of theinvention.

In one embodiment, the method comprises:

-   -   (a) arranging for a sample of cells (e.g. white blood        stem/progenitor cells or biopsy tissue) from a subject to be        tested for the presence of cancer cells expressing IL1RAP above        a threshold criteria using a method according to the sixteenth        or seventeenth aspect of the invention;    -   (b) selecting for treatment subjects whose sample of cells        tested in step (a) contains cancer cells with IL1RAP expression        above a threshold criteria; and    -   (c) administering to the subject selected in step (b) a        therapeutic agent effective in the treatment of said cancer, for        example an antibody polypeptide according to the first aspect of        the invention.

In a related embodiment, the method comprises:

-   -   (a) obtaining a sample of cells (e.g. white blood        stem/progenitor cells or biopsy tissue) from a subject    -   (b) testing said cells for the presence of cancer cells        expressing IL1RAP above a threshold criteria using a method        according to the sixteenth or seventeenth aspect of the        invention;    -   (c) selecting for treatment subjects whose sample of cells        tested in step (b) contains cancer cells with IL1RAP expression        above a threshold criteria; and    -   (d) administering to the subject selected in step (c) a        therapeutic agent effective in the treatment of said cancer, for        example an antibody polypeptide according to the first aspect of        the invention.

Preferences and options for a given aspect, feature or parameter of theinvention should, unless the context indicates otherwise, be regarded ashaving been disclosed in combination with any and all preferences andoptions for all other aspects, features and parameters of the invention.For example, in one embodiment the invention provides an intact IgG1antibody comprising a heavy chain variable region having the amino acidsequence of SEQ ID NO:9 and a light chain variable region having theamino acid sequence of SEQ ID NO:16 for use in the treatment of AML.

The listing or discussion of an apparently prior-published document inthis specification should not necessarily be taken as an acknowledgementthat the document is part of the state of the art or is common generalknowledge.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

These, and other, embodiments of the invention will be betterappreciated and understood when considered in conjunction with the abovedescription and the accompanying drawings. It should be understood,however, that the above description, while indicating variousembodiments of the invention and numerous specific details thereof, isgiven by way of illustration and not of limitation. Many substitutions,modifications, additions and/or rearrangements may be made within thescope of the invention without departing from the spirit thereof, andthe invention includes all such substitutions, modifications, additionsand/or rearrangements.

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Binding of the exemplary antibody in an indirect ELISA to humanIL-1RAP. The exemplary antibody of the invention, CAN04, was found topossess the highest affinity for human IL-1RAP.

FIGS. 2A-C. Binding of exemplary antibody of the invention (CAN04) tohuman CML and AML cells. (2A) The graph shows the MFI value for KU812cells stained with IL1RAP-targeting monoclonal antibodies at aconcentration of 0.1 μg/lmL, and reveals that CAN04 has the highest MFIof the compared antibodies. (2B) CAN04 shows specific binding to fiveprimary AML samples. (2C) In three primary CML samples, CAN04 showsspecific binding.

FIG. 3. Exemplary humanized variants of antibody CAN04 all bind toIL1RAP-expressing BV173 cells, with Variant 6 showing the highest meanfluorescent intensity.

FIGS. 4A-C. Ability of exemplary antibody CAN04 to block (4A) IL-1β (4B)IL-1α, and (4C) IL-33 signalling.

FIG. 5. The total cell expansion of primary primitive CML cells inpresence of IL1b is significantly reduced with exemplary antibody CAN04in comparison with both control and a reference antibody. The graphshows the combined result from three individual patient samplesnormalised to the control consisting of culturing without antibody orwith isotype control antibody.

FIGS. 6A-D. In vitro ADCC assay shows that exemplary antibody CAN04induces specific cell killing of CML cells. (6A) KU812, LAMA84, andBV173 cells were specifically killed by addition of 0.1 μg/mL CAN04.(6B) The cell killing mediated by exemplary antibody CAN04 is dosedependant as shown on BV173 target cells. (6C) Cell killing of primarycells from two CML blast crisis patients was induced by 1 μg/mL CAN04.(6D) Cells from a third CML blast crisis patient carrying the T315Imutation were sensitive to the ADCC effect mediated by CAN04. Eachexperiment was performed at least twice with NK cells from differentdonors, and the presented data shows one representative experiment fromeach.

FIG. 7. In vitro ADCC assay showing that the exemplary CAN04 antibody isefficient in inducing specific cell killing of melanoma cells (SKMEL5cell line). At all concentrations tested, as low as 0.1 μg/mL CAN04shows a high specific killing. The experiment was performed at leasttwice with NK cells from different donors, and the presented data showsone representative experiment.

FIGS. 8A-B. (8A) Confocal images (one optical section, about 0.9 μmthick) showing LAMA cells incubated for 2 hours with CAN04-AF488conjugated antibodies (green) on ice, or at 37° C. for 2 hours, or beenincubated with CAN04-AF488 conjugated antibodies (green) for 16 hours at37 C.°. A clearly defined antibody binding to the cell membrane of themajority of cells can be observed after 2 hours incubation on ice (“Ice2h)”. After incubation for 2 hours with CAN04-AF488 conjugatedantibodies (green) at 37° C., in addition to membrane binding,antibodies have started to enter the cells (internalization) and are nowlocalized also in the cytosol. After 16 hours of incubation at 37° C.,the cell membrane binding is still present and the antibodyinternalization has produced accumulation of CAN04-AF488 antibodies inthe majority of cells. Scale bar (right image) represents 20 μm in allimages. (8B) Control for the CAN04-specific binding and internalization:The confocal images (one optical section, about 0.9 μm thick) show LAMAcells incubated with AF488 conjugated isotype control antibody on ice orat 37° C. for 2 hours, or at 37 C.° for 16 hours. The isotype controlantibody showed no specific binding at any of these conditions. Minorbinding to cellular debris and necrotic cells (weak green) was noted.Scale bar (right image) represents 20 μm in all images.

FIGS. 9A-D. Treatment with CAN04 significantly reduces the leukemiaburden. (9A) The frequency of leukemic cells in peripheral blood waslower in mice treated with exemplary antibody CAN04 compared to isotypecontrol at day 36 after transplantation (1.2% vs. 22.7%, p<0.0001). (9B)The platelet (PLT) count remained at normal levels with CAN04(p=0.0001). (9C) At time of sacrifice the frequency of leukemic cells inthe bone marrow was reduced with CAN04 (38.7% vs. 91.5%; p<0.0001). (9D)The frequency of leukemic cells in spleen was lower in mice treated withCAN04 compared to isotype control (27.6% vs. 70.4%; p=0.0063).

FIGS. 10A-C. The effect of low fucose and Fc mutations on ADCC activity,as determined using SKMEL5 cells. (10A) ADCC activity in SKMEL-5 ofCAN04 (Fc-1) normal fucose, CAN04 (Fc-1) low fucose, Fc-4 and Fc-7.(10B) ADCC activity in SKMEL-5 of CAN04 (Fc-1) normal fucose, CAN04(Fc-1) low fucose, Fc-2 and Fc-3. (10C) ADCC activity in SKMEL-5 ofCAN04 (Fc-1) normal fucose, CAN04 (Fc-1) low fucose, Fc-5, Fc-6 andFc-8.

EXAMPLES

A. Binding Affinity of Exemplary Antibodies of the Invention for IL1-RAPProtein

(i) Biacore Study—Anti-IL1RAP Antibodies of Murine Origin

Materials & Methods

Goat anti-mouse IgG was immobilized on a CM5 chip according to thetechnical manual of capture kit and standard operation principle ofBIAcore T200 (Biacore Life Sciences, GE Healthcare Europe GmbH, Uppsala,Sweden).

The binding analysis cycle consisted of three steps: (i) capture of theligand on the chip surface by immobilized anti-mouse antibody; (ii)binding of the analyte to the captured ligand; and (iii) dissociation ofbound analyte.

The capture molecule surface was regenerated after each binding cycleusing the manufacturer's recommended conditions.

All binding cycles were run at 25° C.

After five cycles of start-up, each antibody (100 nM) was injected at aflow rate of 30 μl/min, for 120 s, at the start of the cycle; then theanalyte (100 nM) was injected at a flow rate of 30 μl/min, for 120 s,followed by monitoring the dissociation phase for 300 s.

One exemplary antibody of the invention (CAN04) was tested along withtwo comparator anti-IL1RAP antibodies (CAN01 and CAN03).

Results & Conclusions

Results are shown in Table 2 below:

TABLE 2 Measurement of K_(on), K_(off) and K_(D) Antibody ka (1/M · s)kd (1/s) KD (M) CAN01 2.34E+05 3.35E−04 1.43E−09 CAN03 2.26E+05 7.25E−053.21E−10 CAN04 4.27E+05 4.72E−05 1.10E−10

The exemplary antibody of the invention, CAN04, exhibited the highestaffinity for human IL1RAP.

(ii) ELISA Study—Anti-IL1RAP Antibodies of Murine Origin

Materials & Methods

An indirect ELISA assay was performed. All samples were analysed induplicate. Nunc-MaxiSorp 96 Micro Well™ Plates were coated with 100 ngof recombinant hIL1RAP 21-367 (100 μl/well) diluted in 0.01M PBS, pH7.4, and incubated overnight at 4° C. Plates were washed with ELISAwashing buffer (0.01M PBS, 0.05% Tween 20, pH 7.4) followed by ablocking step using 150 μl/well of ELISA blocking solution (PBS, 0.5%BSA, 0.05% Tween 20, pH 7.4). After 1 h incubation at room temperature(RT) on agitation the plates were washed again using ELISA washingbuffer. Samples were diluted in three fold serial dilution (ranging from1000 ng/ml to 0.5 ng/ml) in ELISA blocking solution and then transferredto the ELISA plate, 100 μl/well. Plates were incubated at RT for 1 h onagitation and then washed with ELISA washing solution. 100 μl/well ofrabbit anti-mouse IgG conjugated to Alkaline Phosohatase (DAKO, 1:1000)was added and incubated 1 hour at RT on agitation. The plates werewashed followed by addition of substrate (4-Nitrophenyl phosphatisedisodium salt hexahydrate, SIGMA, 1 mg/ml), 100 μl/well. The plates werethereafter incubated at RT on agitation and absorbance at 405 nmmeasured consecutively for 30 min. Absorbance at 0 min was taken asbackground signal.

Results & Conclusions

Results are shown in FIG. 1

The exemplary antibody of the invention, CAN04, was found to possess thehighest binding signal for human IL1RAP.

(iii) ELISA Study—Humanised Versions of the Exemplary ‘CAN04’ Antibody

Materials & Methods

Heavy and light chain variable domains of sixteen humanised variants ofCAN04 (hCAN04) were sub-cloned into vectors containing human constantdomains:

-   -   Kappa constant domain for VL domain [SEQ ID NO:18]    -   IgG1za heavy chain constant for VH domain [SEQ ID NO:19]

Antibodies were transiently expressed in CHOK1SV cells in a volume of200 ml (shaker flask).

Antibodies were purified using Protein A affinity chromatography.

Purified antibodies were analysed by SDS-PAGE and SE-HPLC.

An indirect ELISA assay was performed as described above with thealteration that the samples were diluted in a three-fold serial dilutionstarting at 3.5 nM and run in replicates of four.

Results & Conclusions

Results are shown in Table 3.

TABLE 3 Affinity for hIL1RAP as determined by ELISA Antibody Heavychain* Light Chain** K_(D) (pM) CAN04 (murine) SEQ ID NO: 1 SEQ ID NO: 2 88 ± 2 hCAN04 Variant 1 SEQ ID NO: 8 SEQ ID NO: 15 125 ± 2 hCAN04Variant 2 SEQ ID NO: 9 SEQ ID NO: 15 171 ± 4 hCAN04 Variant 3 SEQ ID NO:10 SEQ ID NO: 15 450 ± 68 hCAN04 Variant 4 SEQ ID NO: 11 SEQ ID NO: 15470 ± 50 hCAN04 Variant 5 SEQ ID NO: 8 SEQ ID NO: 16 135 ± 2 hCAN04Variant 6 SEQ ID NO: 9 SEQ ID NO: 16 173 ± 4 hCAN04 Variant 7 SEQ ID NO:10 SEQ ID NO: 16 526 ± 44 hCAN04 Variant 8 SEQ ID NO: 11 SEQ ID NO: 16402 ± 64 hCAN04 Variant 9 SEQ ID NO: 8 SEQ ID NO: 17 210 ± 4 hCAN04Variant 10 SEQ ID NO: 9 SEQ ID NO: 17 301 ± 5 hCAN04 Variant 11 SEQ IDNO: 10 SEQ ID NO: 17 504 ± 26 hCAN04 Variant 12 SEQ ID NO: 11 SEQ ID NO:17 620 ± 68 *The heavy chain also comprised the IgG1za heavy chainconstant domains [SEQ ID NO: 19]. **The light chain also comprised akappa constant domain [SEQ ID NO: 18].

Four of the sixteen humanised versions of CAN04 showed minimal or nobinding to IL1RAP (data not shown).

B. Binding of Exemplary Antibodies of the Invention to IL1RAP-expressingCells

(i) Flow Cytometry Study—Anti-IL1RAP Antibodies of Murine Origin

Materials & Methods

Chronic myeloid leukemia (CML) cell line KU812 cells were stained withantibodies raised against IL1RAP or a relevant isotype control. Fordetection, a secondary anti-mIg-APC was used.

One exemplary antibody of the invention (CAN04) was tested along withseven comparator anti-IL1RAP antibodies (CAN01, CAN02, CAN03, CAN05,CAN07, CAN08 and CAN09). An isotype negative control antibody was alsoincluded.

For analysis of primary leukemic cells, three CD34-enriched CML patientsamples and five acute myeloid leukemia (AML) patient samples enrichedfor mononuclear cells were stained with CAN04-PE at concentrations of 1μg/mL and 5 μg/mL respectively, or a PE-conjugated isotype control.Cells were analysed using a FACS CANTO flow cytometer (BD).

Results & Conclusions

Staining of IL1RAP-expressing KU812 leukemia cells reveals a higher meanfluorescence intensity (MFI) for CAN04 compared to the isotype controland other comparator antibodies targeting IL1RAP (FIG. 2 A). Labellingof primary cells from five AML and three CML patients using CAN04 resultin staining above the isotype control in flow cytometric analysis (FIG.2 B-C). The present study shows that CAN04 specifically bind IL1RAP witha higher MFI than other tested monoclonal antibodies on a CML cell line,and that CAN04 also bind to primary CML and AML cells.

(ii) Flow Cytometry Study—Humanised Versions of ‘CAN04’ Antibody

Materials & Methods

Chronic myeloid leukemia (CML) cell line BV173 cells were stained with 1μg/mL test antibody or a relevant isotype control. For detection, asecondary anti-hIgG-PE was used. Cells were analysed using a FACS CANTOflow cytometer (BD).

The test antibodies included one chimeric anti-IL1RAP antibody(‘chimary’) and twelve different humanised versions of the exemplaryCAN04 antibody of the invention (‘Var1’ to ‘Var 12’).

Results & Conclusions

Staining of IL1RAP-expressing BV173 leukemia cells shows that thehumanised CAN04 variants stain with different intensity, but all atlevels above the isotype control (see FIG. 3. Humanized variants 1, 2,5, 6, 9 and 10 of CAN04 (see Table 3) exhibit the strongest labelling.

C. Epitope/Domain Mapping of Exemplary Antibodies of the Invention

Materials & Methods

In order to understand where the different antibody clones bind on theIL1RAP, a structural analysis of the protein was performed revealingthat the extracellular part of the receptor could be divided into threedistinct domains hereafter referred to as domains 1, 2 and 3 (D1, D2,D3) (see Wang et al., 2010, Nature Immunology, 11:905-912, thedisclosures of which are incorporated herein by reference). In order todetermine the domain-binding pattern for the different antibody clones,a series of receptor constructs were generated and binding to thesetested in an ELISA assay.

An indirect ELISA assay was performed. All samples were analysed induplicate. Nunc-MaxiSorp 96 Micro Well™ Plates were coated with 100 ngof Rec hIL1 RAP Domain123 (aa21-367) (positive control), Rec hIL1RAPDomain12 (aa21-234), Domain1 (aa21-134) or Rec hIL1RAP Domain3(aa235-367) (100 μl/well) diluted in 0.01M PBS, pH 7.4, and incubatedovernight at 4° C. Plates were washed with ELISA washing buffer (0.01MPBS, 0.05% Tween 20, pH 7.4) followed by a blocking step using 150μl/well of ELISA blocking solution (PBS, 0.5% BSA, 0.05% Tween 20, pH7.4). After 1 h incubation at room temperature (RT) on agitation theplates were washed again using ELISA washing buffer. CAN01, CAN03,CAN04, CAN05, CAN07, CAN08 and KMT-1 (positive control) were diluted inthree fold serial dilution (ranging from 1000 ng/ml to 0.5 ng/ml) inELISA blocking solution and then transferred to the ELISA plate, 100μl/well. Plates were incubated at RT for 1 h on agitation and thenwashed with ELISA washing solution. 100 μl/well of rabbit anti-mouse IgGconjugated to Alkaline Phosphatase (DAKO, 1:1000) was added andincubated 1 hour at RT on agitation. The plates were washed followed byaddition of substrate (4-Nitrophenyl phosphatise disodium salthexahydrate, SIGMA, 1 mg/ml), 100 l/well. The plates were thereafterincubated at RT on agitation and absorbance at 405 nm measuredconsecutively for 30 min. Absorbance at 0 min was taken as backgroundsignal.

One exemplary antibody of the invention (CAN04) was tested along withnine comparator anti-IL1RAP monoclonal antibodies (CAN01, CAN02, CAN03,CAN05, CAN07, CAN08, CAN10, and CAN11, together with a polyclonalanti-IL1RAP antibody (KMT-1) as a positive control.

Results & Conclusions

A majority of anti-IL1RAP antibodies tested for target validation bindto domain 3 (D3). However, the exemplary CAN04 antibody of the inventionis distinct in that it binds to domain 2 (D2). The entire domain mappingdata can be found summarized in the Table 4 below.

TABLE 4 Epitope mapping of exemplary anti-IL1RAP antibody clones.Domain123 Domain12 Domain1 Domain3 Suggested Clone (aa21-367) (aa21-234)(aa21-134) (aa235-367) epitope CAN03 + + D3 CAN05 + + + D1 CAN07 + + D3CAN08 + + D3 CAN04 + + D2 CAN01 + + D3 CAN02 + nd* KMT-1 + + + +polyclonal nd* = not determined as epitope mapping data could notclearly identify specific domain for these constructs, which may beattributed to binding to a structural epitope containing sequenceelements from more than one domain, e.g. D2-D3 junction.D. Specificity/Cross-reactivity of Exemplary Antibodies of the InventionMaterials & Methods

An important feature of a good lead candidate antibody is that itcross-reacts with equal or near-equal potency to the homologous proteinin a relevant toxicology species. According to the general regulatoryguidelines, binding to one rodent and one non-rodent would be thepreferred scenario, but for antibodies this is rarely the case, andinstead many labs struggle to identify any relevant toxicology speciesexcept for primates.

For the present study, cross reactivity to non-human primates likeMacaca mulatta (rhesus) or Macaca fascicularis (cynomolgus) was expectedsince the IL1RAP protein in these species share 99% homology to thehuman IL1RAP protein.

A number of potential lead antibodies were selected and tested forbinding to recombinant M. fascicularis IL1RAP (aa21-367) in an ELISAassay.

One exemplary antibody of the invention (CAN04) was tested along witheight comparator anti-IL1RAP monoclonal antibodies (CAN01, CAN02, CAN03,CAN07, CAN08, CAN09, Mab676 from R&D, and a polyclonal anti-IL1RAPantibody (KMT-1).

Results & Conclusions

Surprisingly, several of the comparator anti-L1 RAP antibodies testedwere found not to cross-react with cynomolgus IL1RAP, amongst them thecommercial reference antibody mAb676 from R&D, Table 5.

TABLE 5 Binding to cynomolgus IL1RAP (Values in bold denotes clonesidentified to cross-react with IL1RAP from M. fascicularis) Binding torec. M. fascicularis Clone IL1RAP (OD₄₀₅) CAN01 0.324 CAN02 0.014 CAN090.022 CAN03 0.870 CAN04 0.416 CAN07 0.111 CAN08 0.375 mAb676 (R&D) 0.037KMT-1 0.481E. Inhibition of IL-1α, IL-1β and IL-33 Signalling by ExemplaryAntibodies of the Invention(i) Effect of CAN04 on IL-1 Signalling in HEK-Blue IL-33/IL-1β Cell LineMaterials & Methods

As IL1RAP is a functional part of the IL-1 receptor complex, antibodiesbinding to IL1RAP may also inhibit IL-1 signalling. Since a number oftumour cell types have been shown to use IL-1 as a growth factor, thismay be an important additional mechanism for mediating anti-tumoureffects.

In order to test for the capability of potential lead candidateantibodies to block IL-1 signalling, an IL-1 dependent reporter geneassay was set up. HEK-Blue IL-33/IL-1β cells (InvivoGen) respond to IL-1signalling by the release of alkaline phosphatase that can be quantifiedby a colorimetric assay. To test the inhibitory capacity of the leadcandidates HEK-Blue cells were plated at 50 000 cells/well and incubatedwith the test antibodies 45 minutes prior to stimulation with IL-1α,IL-1β or IL-33 in a final concentration in assay of 0.3 ng/ml for eachligand. Final assay concentrations of antibodies were 100 nM-0.01 nM. Inthe control wells, the antibodies were replaced by PBS. The cells wereincubated at 37° C. o/n before measuring the amount of alkalinephosphatase released. Antibodies were also tested for potentialagonistic effects by incubating the cells in the presence of a highconcentration of antibody (10 mg/ml) in the absence of additionalstimuli. Any IL-1R agonistic effects would thus be recorded as a releaseof alkaline phosphatase.

One exemplary antibody of the invention (CAN04) was tested along withtwo comparator anti-IL1RAP monoclonal antibodies (CAN01 and CAN 03) andan isotype negative control antibody.

Results & Conclusions

As depicted in FIG. 4(A), the exemplary antibody CAN04 induced apronounced inhibition of IL-1β signalling. Comparator antibody CAN03also produced a detectable inhibition but significantly less than CAN04.Neither the comparator CAN01 nor the isotype control produced anymeasurable inhibition of IL1 signalling.

In addition to blocking IL-1β, CAN04 is also a potent inhibitor of IL-1αand IL-33 signalling; see FIGS. 4(B) and (C), respectively.

None of the tested candidates showed any agonistic effect.

(ii) Effect in Primary CML Cells

Materials & Methods

Bone marrow aspirates or peripheral blood was drawn from three patientswith chronic myeloid leukemia (CML) in chronic phase. Mononuclear cellswere isolated by centrifugation over Lymphoprep, and samples wereenriched for CD34+ cells using an anti-CD34 antibody and magnetic beads(Miltenyi biotech.) The CD34+ cells were kept in liquid nitrogen untiluse, when they were thawed and stained with antibodies against CD34 andCD38. Propidium iodide was used as viability marker. Using a FACS Ariacell sorter, viable CD34+CD38− cells were sorted at a density of 2500cells per well into 96 wells tissue culture treated plates containing100 μl serum free Stemspan culturing medium without supplements. Aftersorting, IL1b and the test antibody were added to the wells in a totalof 100 ul Stemspan to produce a final volume of 200 μl per well with0-0.4 ng/mL IL1b and 0-10 μg/mL antibody. The plates were incubated at37° C., 5% CO₂, for 7 days after which the number of viable (7AAD-)cells were counted using Countbright counting beads and a FACS Canto.

One exemplary antibody of the invention (CAN04) was tested along withone comparator anti-IL1RAP monoclonal antibodies (CAN01) and an isotypenegative control antibody.

Results & Conclusions

As shown in FIG. 5, culturing of CD34+CD38− primary chronic phase CMLcells in the presence of IL1b results in increased cell expansion. TheIL1-induced increase in cell expansion is significantly reduced uponaddition of the exemplary CAN04 antibody to the culture (p<0.0001). Alsoin comparison with another IL1RAP-targeting antibody, CAN01, CAN04 issignificantly more effective in reducing the total cell expansion(p=0.0022). We conclude that binding of CAN04 to IL1RAP interferes withthe stimulation in cellular expansion induced by IL1 in primaryprimitive CML cells.

F. ADCC Effect of Exemplary Antibodies of the Invention

(i) Chronic Myeloid Leukemia (CML) Cell Lines

Materials & Methods

Chronic myeloid leukemia (CML) cell lines KU812, LAMA84 and BV173, orprimary cells from three patients with CML in blast crisis were used astarget cells in the in vitro antibody dependent cellular cytotoxicity(ADCC) assay. Briefly, target cells were labelled with PKH26(Sigma-Aldrich, St Louis, Mo.) according to manufacturer's instructions,and seeded into a 96-well plate at a density of 5,000-10,000 cells perwell. Subsequently, the exemplar antibody of the invention, CAN04, orisotype control antibody was added to wells in different concentrationsand incubated for 30 min before 100,000 NK effector cells were added toeach well. NK-cells were extracted from healthy volunteers afterinformed consent by using an NK-cell negative cell isolation kitaccording to manufacturer's instructions (Miltenyi Biotech, BergischGladbach, Germany). A non-specific human IgG1 antibody was used as anisotype negative control in the experiments (Eureka Therapeutics,Emeryville, Calif.). The degree of cell death was assessed by detectionof 7-AAD positive cells using a FACS CANTO flow cytometer (BD). Eachexperiment was performed at least twice with NK cells from differentdonors.

Results & Conclusions

The in vitro ADCC assay shows that the exemplary antibody of theinvention, CAN04, directs NK-cells to kill CML cell lines KU812, LAMA84and BV173 to a higher degree than the isotype control (FIG. 6A). A dosetitration of CAN04 using BV173 target cells shows that the effect oncell killing is dose dependent with a higher degree of cell killing withincreasing CAN04 concentration (FIG. 6B). Chronic myeloid leukemia thathas progressed into blast crisis display only transient effect totreatment with tyrosine kinase inhibitors and thus imposes a majortreatment problem. The ADCC assay with primary cells from two individualCML blast crisis patients shows that these cells were sensitive to thecellular cytotoxicity induced by CAN04 and NK-cells (FIG. 6C). Inaddition, primary cells from a third CML blast crisis patient harbouringthe T315I mutation that cause resistance to several tyrosine kinaseinhibitors display similar sensitivity (FIG. 6D). Altogether, theexperiments show that CAN04 has the ability to direct NK-cells tospecific cell killing of CML cell lines as well as primary blast crisisCML cells, and that the cytotoxic effect induced by CAN04 is dosedependent.

(ii) Melanoma Cell Lines

Materials & Methods

The malign melanoma cell line SKMEL-5 was used as a target for in vitroantibody dependent cellular cytotoxicity (ADCC) assay. Briefly, targetcells were labelled with PKH26 (Sigma-Aldrich, St Louis, Mo.) accordingto manufacturer's instructions, and seeded into a 96-well plate at adensity of 5,000-10,000 cells per well. Subsequently, CAN04 or isotypecontrol antibody were added to wells in different concentrations andincubated for 30 min before 100,000 NK effector cells were added to eachwell. NK-cells were extracted from healthy volunteers after informedconsent by using an NK-cell negative cell isolation kit according tomanufacturer's instructions (Miltenyi Biotech, Bergisch Gladbach,Germany). A non-specific human IgG1 antibody was used as control in theexperiments (Eureka Therapeutics, Emeryville, Calif.). The degree ofcell death was assessed by detection of 7-AAD positive cells using aFACS CANTO flow cytometer (BD). Each experiment was performed at leasttwice with NK cells from different donors.

Results & Conclusions

The in vitro ADCC assay showed that CAN04 directs NK-cells to killing ofthe SKMEL-5 cell line to a much higher degree than a matching isotypecontrol (FIG. 7). The dose titration of CAN04 showed that CAN04 isefficient in inducing ADCC, at low concentrations (FIG. 7). In summary,these data demonstrate that CAN04 has the ability to direct NK-cells tospecific cell killing of SKMEL-5 in a dose dependent manner and thatCAN04.

G. Internalisation of Exemplary Antibodies of the Invention

Materials & Methods

Cells and Culture Conditions: LAMA-84 cells, a cell-line establishedfrom a patient with chronic myeloid leukemia in blast crisis, wereobtained from DSMZ (Braunschweig, Germany) and cultured according to therecommendation by the supplier. Briefly, cells were cultured in RPMI1640 with 10% FBS, 1% Glutamine and 1% Penicillin/Streptomycin in 5%CO₂, 37° C. Cell cultures were split to a density of 0.5×10⁶ cells/mlevery 2-3 days. Cells were used for up to 12 passages after they werereceived from DSMZ.

Cells from the suspension cell-line LAMA-84 were washed once inphosphate buffered saline (PBS) supplemented with 1% Bovine serumalbumin (BSA) and resuspended in PBS-BSA supplemented with 5% human AB+serum from Sigma and incubated for 5 minutes at room temperature (RT).The AlexaFluor488 (AF488) labelled IL-1RAP selective antibody CAN04, orisotype matched control antibody, was added to a final concentration of10 μg/ml. Cells were placed (incubated) on ice or at 37° C. for 2 or 16hours.

For the image analysis with confocal microscopy (LSM 510 Meta Zeissconfocal microscope), cells were washed twice in PBS-1% BSA, werebriefly spun down followed by resuspension in 3% paraformaldehyde (inPBS) fixation for 20 minutes (at 4 C.°). Cells were then spun down,resuspended in PBS containing 0.001% Triton X-100 (PBS-TX) and a nuclearmarker (DAPI), and were let to incubate for 5 minutes at RT. After abrief centrifugation cells were resuspended in PBS-TX and placed inglass-bottomed microscope wells. Cells were then let to adhere for onehour. Image data were collected via confocal scanning of cells providinghigh-resolution images of AlexaFluor488 fluorescence in thin opticalsections through the centre of cells (depicted by nuclear marker).Further analyses of antibody binding to cell membrane and/orinternalized antibodies were performed via software image analyses(Zeiss Zen2010).

Results & Conclusions

The structural relation of CAN04 binding to the cell membrane and itscapacity to enter the cells (“internalize”) was demonstrated with highresolution imaging data recorded by means of confocal laser scanningmicroscopy, and by image analyses of this data.

Image data representations are shown in FIG. 8.

H. Therapeutic Efficacy In Vivo of an Exemplary Antibody of theInvention

Materials & Methods

Unconditioned NOD/SCID mice were engrafted with lethal doses of MA9Rascells, previously generated by transformation of human umbilical cordblood CD34⁺ cells by retroviral integration of cDNAs directing theexpression of an MLL/AF9 fusion and an activated NRAS gene. Leukemicmice were treated with the exemplary CAN04 antibody targeting IL1RAP, ora corresponding isotype control antibody. The antibodies wereadministered by intraperitoneal injections twice weekly throughout theexperiment with first treatment given day three after transplantation.Each dose of antibody was 500 μg, except for the first that was given asa bolus of 1000 μg. Mice were sacrificed upon signs of severe disease asjudged by hunchback, untidy fur, and decreased mobility, or due to solidtumours.

Results & Conclusions

Immunodeficient mice were engrafted with human leukemic cells andtreated with CAN04, a monoclonal antibody targeting IL1RAP. Thefrequency of leukemic cells in peripheral blood was significantlyreduced at day 36 after transplantation, and the platelet countsremained normal in mice given CAN04 compared to isotype control antibodyindicating a more functional haematopoiesis (FIG. 9A-B). CAN04 treatmentresulted in a significant reduction of leukemic cells in bone marrow andspleen (FIG. 9C-D). We conclude that anti-IL1RAP immunotherapy reduceshuman leukemia in peripheral blood, bone marrow, and spleen, in theMA9Ras xenograft model. The results support anti-IL1RAP immunotherapy asa new promising therapeutic strategy for AML.

I. Effect of (a) Low Fucose Antibodies and (b) Fc Mutations on ADCCActivity

Materials & Methods

The humanised heavy chain variable domain variant of CAN04 (comprisingSEQ ID No: 9) was sub-cloned into vector containing the of IgG1za humanconstant domain (SEQ ID NO:19).

The humanised light chain variable domain variant of CAN04 (comprisingSEQ ID No: 16) was sub-cloned into vector containing the Kappa humanconstant domain (SEQ ID NO:18).

To examine the effect of low fucose, hCAN04 antibody “Fc-1” (see below)were transiently expressed by co-transfection of the resulting vectorsinto HEK293 cells cultured in medium containing Kinfunensine. AntibodyFc-1, produced by expression in the absence of Kinfunensine, was used asthe ‘Normal Fucose’ control

To address how various Fc-engineered mutants of hCAN04 affect the ADCCactivity, genetically engineered hCAN04 variants were generated asfollows:

-   -   Fc-1=(Humanised CAN04 with wildtype Fc; i.e. hCAN04 variant 6 in        Table 3)    -   Fc-2=(As Fc-1 but with mutations S239D/S298A/I332E)    -   Fc-3=(As Fc-1 but with mutations S239D/A330L/I332E)    -   Fc-4=(As Fc-1 but with mutations S239D/I332E)    -   Fc-5=(As Fc-1 but with mutations S298A/E333A/K334A)    -   Fc-6=(As Fc-1 but with mutation N297Q)    -   Fc-7=(As Fc-1 but with mutation N297S)    -   Fc-8=(As Fc-1 but with mutations P247I/A339Q)

wherein the position of the amino acid mutations is defined using the EuNumbering Scheme, which differs from the numbering in SEQ ID NOS: 18 and19 above; see

Edelman et al., 1969, Proc. Natl. Acad. Sci. USA, 63:78-85).

Antibodies were purified using Protein A affinity chromatography.

ADCC assays were performed using the SKMEL5 cell line.

Results & Conclusions

A low fucose variant of hCAN04, produced in the presence ofkinfunensine, was superior to a normal fucose CAN04-variant (FIG. 10).

The effect of Fc mutation on ADCC activity was dependent upon themutation(s) made. Thus:

-   -   (a) ADCC-activity of the Fc-6 and Fc-7 variants of humanised        CAN04 was completely abrogated;    -   (b) ADCC-activity of the Fc-3, Fc-4, and Fc-5 variants of        humanised CAN04 was enhanced, even relative to the low fucose        variant of CAN04;    -   (c) ADCC-activity of the Fc-8 variant of humanised CAN04 was        similar to that of the low fucose form of CAN04    -   (d) ADCC-activity of the Fc-2 variant of humanised CAN04 was        similar to that of the normal fucose form of CAN04.        Example J—Analysis of Competitive Binding by ELISA        Protocol    -   All samples should be analysed in duplicate.    -   Coat a Nunc-MaxiSorp 96 Micro Well™ Plate with 100 ul/well of        recombinant hIL1RAP 21-367 (1 ug/ml) diluted in 0.01M PBS, pH        7.4.    -   Incubate the plate overnight at 4° C.    -   Wash the plate with ELISA washing buffer        -   (0.01M PBS, 0.05% Tween 20, pH 7.4).    -   Add 150 μl/well of ELISA blocking solution        -   (PBS, 0.5% BSA, 0.05% Tween 20, pH 7.4).    -   Incubate the plate for 1 h at room temperature (RT) under        agitation.    -   Wash the plate with ELISA washing buffer.    -   Add samples of test items (e.g. mAb 1, mAb 2) to wells (100        ul/well, 10 ug/ml)    -   Incubate the plate for 1 h at RT.    -   Wash the plate with ELISA washing solution.    -   Add a solution of reference antibody CAN04 (100 ul/well, 1        ug/ml) to all wells.    -   Incubate the plate for 1 h at RT.    -   Wash the plate with ELISA washing buffer.    -   Add 100 μl/well of a suitable secondary antibody conjugated to        Alkaline rabbit anti-mouse IgG conjugated to Alkaline        Phosphatase (If the test items are human antibodies, a suitable        secondary antibody would be Goat Anti-Mouse IgG (Fc        specific)-Alkaline Phosphatase antibody, SIGMA, A1418)    -   Incubate the plate for 1 h at RT under agitation.    -   Wash the plate with washing buffer.    -   Add 100 μl of pNPP substrate per well.        -   (4-Nitrophenyl phosphatise disodium salt hexahydrate, SIGMA,            1 mg/ml).    -   Incubate the plate at RT under agitation and measure absorbance        at 405 nm consecutively for 30 min. Absorbance at 0 min should        be taken as background signal.

The invention claimed is:
 1. A method for the treatment of a neoplasticdisorder in a subject, comprising administering to the subject aneffective amount of an antibody polypeptide that has binding specificityfor human interleukin-1 receptor accessory protein (IL1RAP), wherein theneoplastic disorder is associated with cells expressing IL1RAP, andwherein the antibody polypeptide comprises a heavy chain variable region(VH) comprising: a. (i) heavy chain CDR1 comprising: GYAFTSS (aminoacids 26-32 of SEQ ID NO:9); (ii) Heavy chain CDR2 comprising: YPGDGN(SEQ ID NO:4); and (iii) Heavy chain CDR3 comprising: GYLDPMDY (SEQ IDNO: 5); and a light chain variable region (VL) comprising: (i) Lightchain CDR 1 comprising: ASQGINNYLN (amino acids 25-34 of SEQ ID NO:16);(ii) Light chain CDR2 comprising: YTSGLHA (SEQ ID NO: 13); and (iii)Light chain CDR3 comprising: QQYSILPWT (SEQ ID NO: 14); b. A heavy chainvariable region (VH) comprising (i) heavy chain CDR1 comprising GYAFTSS(amino acids 26-32 of SEQ ID NO:9); (ii) heavy chain CDR2 comprisingYPGDGN (SEQ ID NO: 4); and (iii) heavy chain CDR3 comprising GYLDPMDY(SEQ ID NO: 5); and a light chain variable region (VL) comprising (i)light chain CDR 1 comprising SASQGINNYLN (SEQ ID NO:12); light chainCDR2 comprising YTSGLHA (SEQ ID NO: 13); and (iii) light chain CDR3comprising QQYSILPWT (SEQ ID NO: 14); c. A heavy chain variable region(VH) comprising (i) heavy chain CDR1 comprising GYAFSSS (SEQ ID NO:3);(ii) heavy chain CDR2 comprising YPGDGN (SEQ ID NO: 4); and (iii) heavychain CDR3 comprising GYLDPMDY (SEQ ID NO: 5); and a light chainvariable region (VL) comprising (i) light chain CDR 1 comprisingSASQGINNYLN (SEQ ID NO:12); light chain CDR2 comprising YTSGLHA (SEQ IDNO: 13); and (iii) light chain CDR3 comprising QQYSILPWT (SEQ ID NO:14); d. A heavy chain variable region (VH) comprising (i) heavy chainCDR1 comprising GYAFSSS (SEQ ID NO:3); (ii) heavy chain CDR2 comprisingYPGDGN (SEQ ID NO: 4); and (iii) heavy chain CDR3 comprising GYLDPMDY(SEQ ID NO: 5); and a light chain variable region (VL) comprising (i)light chain CDR 1 comprising ASQGINNYLN (amino acids 25-34 of SEQ IDNO:16); light chain CDR2 comprising YTSGLHA (SEQ ID NO: 13); and (iii)light chain CDR3 comprising QQYSILPWT (SEQ ID NO: 14); e. A heavy chainvariable region (VH) comprising (i) heavy chain CDR1 comprising GYTFTSS(amino acids 26-32 of SEQ ID NO:10); (ii) heavy chain CDR2 comprisingYPGDGQ (amino acids 52-57 of SEQ ID NO:10); and (iii) heavy chain CDR3comprising GYLDPMDY (SEQ ID NO: 5); and a light chain variable region(VL) comprising (i) light chain CDR 1 comprising SASQGINNYLN (SEQ IDNO:12); light chain CDR2 comprising YTSGLHA (SEQ ID NO: 13); and (iii)light chain CDR3 comprising QQYSILPWT (SEQ ID NO: 14); or f. A heavychain variable region (VH) comprising (i) heavy chain CDR1 comprisingGYTFTSS (amino acids 26-32 of SEQ ID NO:10); (ii) heavy chain CDR2comprising YPGDGQ (amino acids 52-57 of SEQ ID NO:10); and (iii) heavychain CDR3 comprising GYLDPMDY (SEQ ID NO: 5); and a light chainvariable region (VL) comprising (i) light chain CDR 1 comprisingASQGINNYLN (amino acids 25-34 of SEQ ID NO:16); light chain CDR2comprising YTSGLHA (SEQ ID NO: 13 ); and (iii) light chain CDR3comprising QQYSILPWT (SEQ ID NO: 14).
 2. The method of claim 1, whereinthe neoplastic disorder is a neoplastic hematologic disorder.
 3. Themethod of claim 2, wherein the neoplastic hematologic disorder isselected from chronic myeloid leukemia (CML), myeloproliferativedisorders (MPD), myelodysplastic syndrome (MDS), acute lymphoblasticleukemia (ALL) and acute myeloid leukemia (AML).
 4. The method of claim1, wherein the neoplastic disorder is associated with the formation ofsolid tumors within the subject's body.
 5. The method of claim 4,wherein the solid tumour is selected from prostate cancer, breastcancer, lung cancer, colorectal cancer, melanomas, bladder cancer,brain/CNS cancer, cervical cancer, oesophageal cancer, gastric cancer,head/neck cancer, kidney cancer, liver cancer, lymphomas, ovariancancer, pancreatic cancer, and sarcomas.
 6. The method of claim 1,wherein the antibody polypeptide comprises a heavy chain variable regioncomprising or consisting of the amino acids of SEQ ID NO:
 9. 7. Themethod of claim 1, wherein the antibody polypeptide comprises a lightchain variable region comprising or consisting of the amino acids of SEQID NO:
 16. 8. The method of claim 1, wherein the antibody polypeptidecomprises a heavy chain variable region comprising or consisting of theamino acids of SEQ ID NO: 9, and a light chain variable regioncomprising or consisting of the amino acids of SEQ ID NO:
 16. 9. Themethod of claim 1, wherein the antibody polypeptide comprises: a) aheavy chain variable region which comprises or consists of the aminoacid sequence of SEQ ID NO: 8 and a light chain variable region whichcomprises or consists of the amino acid sequence of SEQ ID NO: 15; b) aheavy chain variable region which comprises or consists of the aminoacid sequence of SEQ ID NO: 9 and a light chain variable region whichcomprises or consists of the amino acid sequence of SEQ ID NO: 15; c) aheavy chain variable region which comprises or consists of the aminoacid sequence of SEQ ID NO: 10 and a light chain variable region whichcomprises or consists of the amino acid sequence of SEQ ID NO: 15; d) aheavy chain variable region which comprises or consists of the aminoacid sequence of SEQ ID NO: 11 and a light chain variable region whichcomprises or consists of the amino acid sequence of SEQ ID NO: 15; e) aheavy chain variable region which comprises or consists of the aminoacid sequence of SEQ ID NO: 8 and a light chain variable region whichcomprises or consists of the amino acid sequence of SEQ ID NO: 16; f) aheavy chain variable region which comprises or consists of the aminoacid sequence of SEQ ID NO: 9 and a light chain variable region whichcomprises or consists of the amino acid sequence of SEQ ID NO: 16; g) aheavy chain variable region which comprises or consists of the aminoacid sequence of SEQ ID NO: 10 and a light chain variable region whichcomprises or consists of the amino acid sequence of SEQ ID NO: 16; h) aheavy chain variable region which comprises or consists of the aminoacid sequence of SEQ ID NO: 11 and a light chain variable region whichcomprises or consists of the amino acid sequence of SEQ ID NO: 16; i) aheavy chain variable region which comprises or consists of the aminoacid sequence of SEQ ID NO: 8 and a light chain variable region whichcomprises or consists of the amino acid sequence of SEQ ID NO: 17; j) aheavy chain variable region which comprises or consists of the aminoacid sequence of SEQ ID NO: 9 and a light chain variable region whichcomprises or consists of the amino acid sequence of SEQ ID NO: 17; k) aheavy chain variable region which comprises or consists of the aminoacid sequence of SEQ ID NO: 10 and a light chain variable region whichcomprises or consists of the amino acid sequence of SEQ ID NO: 17; or l)a heavy chain variable region which comprises or consists of the aminoacid sequence of SEQ ID NO: 11 and a light chain variable region whichcomprises or consists of the amino acid sequence of SEQ ID NO:
 17. 10.The method of claim 1, wherein the antibody polypeptide comprises aheavy and light chain constant region, or part thereof.
 11. The methodof claim 1, wherein the antibody polypeptide comprises an Fc region. 12.The method of claim 1, wherein the antibody polypeptide is humanised.13. The method of claim 1, wherein the antibody polypeptide furthercomprises a cytotoxic moiety.
 14. The method of claim 1, wherein theantibody polypeptide further comprises a detectable moiety.
 15. Themethod of claim 1, wherein the antibody polypeptide further comprises amoiety for increasing the half-life of the antibody polypeptide.